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  • 93
    Thermo Fisher anti oct3 4
    Anti Oct3 4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Becton Dickinson anti oct3 oct4
    GP130 Overexpressing Cell Lines Show Non-Monotonic Response of Stat3 Activation to Receptor Overexpression (A) The model-predicted surface of Stat3 activation is shown as a function of time and change in production of GP130 receptors. Successive increase in GP130 production as shown by black, green, and red arrows in that order shows a non-monotonic response of Stat3 activation and the presence of a local maximum. This is illustrated by the circle in the contour map on the x–y plane. (B) GP130 overexpressing cell lines RG1–RG5 were developed to express the receptor at varying levels compared with R1 cells, and these cell lines retain <t>Oct4</t> expression in the presence of LIF (C). (D) Model-predicted trends of GP130 overexpression corresponding to (A) shows normal profile of Stat3 activation (black line), Stat3 activation in slight GP130 overexpression (green line), and significant GP130 overexpression (red line). (E) Experimental results show a consistent trend in LIF-induced Stat3 activation profile using RG1 and RG5 cell lines in comparison with model results in (D). (F) IL-6 stimulation of GP130 overexpressing cell lines shows a dose-dependent increase in Stat3 activation as a function of GP130 overexpression.
    Anti Oct3 Oct4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson oct4
    Contribution of rat EG cells to embryonic chimaeras including germ cells. ( A ) Phase contrast and fluorescence images of Red1.2 EG cell clone. ( B ) Graph showing chromosome number per cell in 50 metaphase spreads from line Red1.2. Inset is a karyotype of Red1.2, showing 42 chromosomes. ( C ) Bright-field and fluorescence images of E13.5 chimaeric embryos. ( D ) Confocal section of genital ridge and surrounding mesonephric tissue dissected from a chimaeric embryo and immunostained for DsRed and <t>Oct4.</t> ( E ) Higher magnification confocal section of D shows cells co-stained for DsRed and Oct4, demonstrating the contribution of EG cells to germ cells. Scale bars: 100 μm in A,D; 25 μm in E.
    Oct4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 239 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology oct3 4 pou5f1
    FGF4 and heparin rescue neural differentiation of NDST1 −/− NDST2 −/− ES cells. A , the addition of conditioned medium from WT ES cells subjected to neural differentiation to the NDST1 −/− NDST2 −/− cells rescued neural differentiation of the mutant cells ( top , day 13). The addition of 10 ng/ml FGF4 only for 7 days had no effect on mutant cells in N2B27 ( middle ), whereas 10 ng/ml FGF4 in combination with 0.1 μg/ml heparin rescued neural differentiation ( bottom , day 9). B , immunofluorescence staining of mutant cells. Shown is neural differentiation in the presence of FGF4 and heparin ( top ) and staining for nestin ( red ) and DAPI ( blue ). Mutant neural cells were plated on a polyornithine/fibronectin-coated surface 5 days after withdrawal of FGF4 and heparin and stained for the neuronal marker Tuj1 ( red ), the astrocyte marker GFAP ( green ), and DAPI ( blue ). C , quantification of <t>Oct3/4</t> and nestin-expressing NDST1 −/− NDST2 −/− cells subjected to 6 days of neural differentiation in N2B27 medium with and without the addition of FGF4 and heparin. Error bars , S.D.
    Oct3 4 Pou5f1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    STEMCELL Technologies Inc anti oct3 4
    FGF4 and heparin rescue neural differentiation of NDST1 −/− NDST2 −/− ES cells. A , the addition of conditioned medium from WT ES cells subjected to neural differentiation to the NDST1 −/− NDST2 −/− cells rescued neural differentiation of the mutant cells ( top , day 13). The addition of 10 ng/ml FGF4 only for 7 days had no effect on mutant cells in N2B27 ( middle ), whereas 10 ng/ml FGF4 in combination with 0.1 μg/ml heparin rescued neural differentiation ( bottom , day 9). B , immunofluorescence staining of mutant cells. Shown is neural differentiation in the presence of FGF4 and heparin ( top ) and staining for nestin ( red ) and DAPI ( blue ). Mutant neural cells were plated on a polyornithine/fibronectin-coated surface 5 days after withdrawal of FGF4 and heparin and stained for the neuronal marker Tuj1 ( red ), the astrocyte marker GFAP ( green ), and DAPI ( blue ). C , quantification of <t>Oct3/4</t> and nestin-expressing NDST1 −/− NDST2 −/− cells subjected to 6 days of neural differentiation in N2B27 medium with and without the addition of FGF4 and heparin. Error bars , S.D.
    Anti Oct3 4, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam oct3 4
    Characterization of mesenchymal stem cells. Primary human mesenchymal stem cells immunostained for <t>Oct3/4</t> (A), and Nanog (B). Cells were fixed and permeabilized. Primary antibodies were detected by addition of fluorescein-labelled (FITC) (green) and Alexa Fluor 594-labelled (red) secondary antibodies. E, G-I: Double immunofluorescence staining for CD90 (E), CD105 (G, H, I) and C-X-C-chemokine receptor type 4 (CXCR4) (panels E, G, H, I) in lung tissue sections from patients with histologically confirmed IPF/UIP and chronic fibrotic hypersensitivity pneumonitis. Primary antibodies were detected by addition of secondary antibodies labelled with fluorescein FITC (green) for CD105 and CXCR4 detection or Cy3-labelled (red) for CD90 and CXCR4 detection. Magnification x40. Images were acquired using LSM 510 confocal microscope. Panel H and I is a 3D reconstruction to show more clear co stainings on the same cell. F: Co-staining with CD44 (pink) and CXCR4 (dark red) (Scale bar 2μm, Magnification x100 (oil)). (C, D) Oct3/4 (C) and Nanog (D) mRNA expression in mesenchymal stem cells (black bars) and in fibroblasts (grey bars). RNA expression was assessed by quantitative real time RT-PCR. Data are presented as mean ± SEM of independent experiments performed in three different cell lines.
    Oct3 4, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher oct3
    A, quantification of hOCT3 and hPMAT mRNA levels in stably transfected Flp-in HEK293 cells. Total RNA from Flp-293-pcDNA5, hOCT3, and hPMAT cells were extracted and reverse-transcribed. hOCT3 and hPMAT mRNA copy numbers were determined by Taqman real-time RT-PCR using specific primers and probes. Diluted hOCT3 and hPMAT expression vectors with known copy numbers were used as standards to determine absolute mRNA copy numbers. B, localization of hOCT3 and hPMAT in Flp-in HEK293 cells stably transfected with hPMAT or hOCT3. Cells transfected with pcDNA5 vector were used as control. Confluent cells were immunostained with <t>anti-OCT3</t> or anti-PMAT primary antibodies and Alexa Fluor-conjugated secondary antibodies. Images were taken under a fluorescent microscope with corresponding filters.
    Oct3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend oct3 4
    A, quantification of hOCT3 and hPMAT mRNA levels in stably transfected Flp-in HEK293 cells. Total RNA from Flp-293-pcDNA5, hOCT3, and hPMAT cells were extracted and reverse-transcribed. hOCT3 and hPMAT mRNA copy numbers were determined by Taqman real-time RT-PCR using specific primers and probes. Diluted hOCT3 and hPMAT expression vectors with known copy numbers were used as standards to determine absolute mRNA copy numbers. B, localization of hOCT3 and hPMAT in Flp-in HEK293 cells stably transfected with hPMAT or hOCT3. Cells transfected with pcDNA5 vector were used as control. Confluent cells were immunostained with <t>anti-OCT3</t> or anti-PMAT primary antibodies and Alexa Fluor-conjugated secondary antibodies. Images were taken under a fluorescent microscope with corresponding filters.
    Oct3 4, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore oct3 4
    TESC regulates the CSC-like self-renewal properties in lung cancer cells. ( A ) Clonogenicities of TESC -knockdown A549 cells with siRNA and TESC -overexpressing H460 cells transfected with expression vector ( TESC -pcDNA3.1) were examined in vitro by colony formation assay. ( B ) Cellular levels of TESC and CSC markers including CD44, CD133, <t>Oct3/4,</t> Sox2, and β-catenin in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( C ) RT-PCR analysis of ALDH1 isozymes in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( D ) Changes in sphere-forming capacity in TESC -knockdown A549 cells or TESC -overexpressing H460 cells. ( E ) Changes in cellular ALDH1 levels were examined using ALDEFLUOR assay in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. Data represent mean ± SD of three independent experiments using two-tailed t-test. *P
    Oct3 4, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc oct3 4
    Effect of Cltc Knockdown in mESCs Can Be Partially Rescued by Inhibiting TGF-β Signaling (A) Bright-field micrographs showing AP staining and morphology of Cltc knockdown mESCs in the presence of TGF-β inhibitors RepSox or SB431542 (SB). Scale bar, 50 μm. (B) Immunofluorescence micrographs showing <t>OCT3/4</t> and E-CAD staining upon RepSox or SB431542 (SB) treatment in mESCs with Cltc knockdown. Scale bar, 50 μm. (C) Bar graph showing the quantitation of OCT3/4 and E-CAD-positive mESC colonies upon RepSox or SB431542 (SB) treatment in Cltc knockdown mESCs. Error bars represent mean ± SD from three independent experiments (n = 3). ∗∗ p
    Oct3 4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stemgent oct3 4
    Effect of Cltc Knockdown in mESCs Can Be Partially Rescued by Inhibiting TGF-β Signaling (A) Bright-field micrographs showing AP staining and morphology of Cltc knockdown mESCs in the presence of TGF-β inhibitors RepSox or SB431542 (SB). Scale bar, 50 μm. (B) Immunofluorescence micrographs showing <t>OCT3/4</t> and E-CAD staining upon RepSox or SB431542 (SB) treatment in mESCs with Cltc knockdown. Scale bar, 50 μm. (C) Bar graph showing the quantitation of OCT3/4 and E-CAD-positive mESC colonies upon RepSox or SB431542 (SB) treatment in Cltc knockdown mESCs. Error bars represent mean ± SD from three independent experiments (n = 3). ∗∗ p
    Oct3 4, supplied by Stemgent, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Santa Cruz Biotechnology oct3 4a
    Generation of human reprogrammed cells from fibroblasts of control healthy subjects and HNF1A MODY patients. (A). Morphology of cells from healthy subject before (day 0) and 13 days after transduction (day 13) with hSTEMCCA lentiviral vectors. Colonies resembling human embryonic stem cells, visible at day 13, were further expanded (expanded colony, shown at passage 2). GFP - expression of GFP in human primary fibroblast isolated from HNF1A MODY patients, 72 after transduction with control (FUGW) lentiviral vectors. Representative pictures (scale bar – 100 μm). (B). Analysis of hSTEMCCA construct silencing in established control (effective silencing in Ctr Repro 2 cell line) and HNF1A MODY reprogrammed cells (effective silencing in HNF1A MODY Repro 1 cell line). RT-PCR. (C,D). Immunofluorescence staining of pluripotency markers: <t>OCT3/4A,</t> SSEA4, TRA-1-60, TRA-1-81 in control reprogrammed cells (C) and HNF1A MODY reprogrammed cells (D). Representative pictures (scale bar – 100 μm).
    Oct3 4a, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher oct3 4
    LSD1 is minimally expressed in hiPSCs but strongly expressed in hiPSC-derived teratoma (A) We isolated whole cell lysates from normal human fibroblasts, hiPSC line 201B, 201B-derived teratoma, normal human T-lymphocytes, and cancer cell lines (K562, HeLa, and HEK293) for immunoblot analyses to determine the expression of HDAC1, HDAC3, HDAC6, LSD1, <t>Oct3/4,</t> and GAPDH (internal control) (upper panel). The signal intensities of each band were quantified, normalized to those of the corresponding GAPDH, and shown as relative values setting fibroblast at 1.0 (lower panel). Immunoblotting was carried out using the Simple Western System Wes. (B) We isolated total cellular RNAs and subjected them to qPCR to evaluate the expression of HDAC1, HDAC3, HDAC6 , and LSD1 mRNA. Data were quantified by the 2 –ΔΔCt method using simultaneously amplified GAPDH as a reference and are shown as relative values setting fibroblast at 1.0. (C) Frozen continuous sections were prepared from the developed teratomas and subjected to hematoxylin-eosin (HE) and immunofluorescent chemical (IFC) staining. IFC specimens were stained with anti-HDAC1, anti-HDAC6, or anti-LSD1 antibodies, followed by staining with Alexa Fluor 488-conjugated anti-rabbit IgG (green). Nuclei were counterstained with DAPI (blue). Only merged images are shown. Scale bars indicate 200 μm (upper panels) and 50 μm (lower panels), respectively. Data shown are representative of multiple independent experiments.
    Oct3 4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 305 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend pe anti oct4 oct3
    Metformin decreases K7M2 xenograft tumor growth and inhibits lung metastasis in vivo . (a) Metformin suppressed tumorigenicity of K7M2 OSCs in vivo by limited dilution assay. n = 7. (b) The tumor formation rate of K7M2 OSCs treated with metformin. (c) The tumor volumes and (d) tumor weights at the end of the experiments. (e) Images of resected tumor xenografts on day 21. n = 6. (f) The tumor volumes and (g) the tumor weights at the end of the experiments. (h) Numbers of mice with lung metastasis. (i) H E staining of lung tissues for metastatic nodules. The arrows represent smaller tumor nodules in the lung. Scale bars = 100 μ m. (j) Immunohistochemical staining of LC3, ATG5, Ki67, Sox2, and <t>Oct4</t> of three different tumors from each group. Scale bars = 100 μ m. ∗ P
    Pe Anti Oct4 Oct3, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    R&D Systems anti oct3 4
    ECFC-derived iPSCs express typical hESC markers. (A) Immunofluorescence analysis of ECFC-derived iPSC1 line. Expression of stem cell markers: <t>OCT3/4,</t> SOX2, NANOG, PA, SSEA4, TRA-1-81. The cells were stained using the 488 and 546-conjugated Donkey Anti-Goat IgG or Goat Anti-Mouse IgG secondary antibody and the nuclei were counterstained with DAPI (blue). Scale bars represent 100 μm. (B) Quantitative RT-PCR analysis of the expression of endogenous (endo) and exogenous (exo) markers: OCT3/4 , SOX2 , KLF4 and C-MYC in Ctrl ECFCs, transduced ECFCs (ECFC OKSM) and ECFC-derived iPSC1 at passage 4, 7 and 10. Transcript levels were normalized to GAPDH transcript levels and relative to mean hESCs (H9 samples at P45) as a calibrator.
    Anti Oct3 4, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ABclonal anti oct4
    1α,25(OH)2D3 inhibited the stemness and impaired the mitochondrial respiration and ATP production in acidic condition. a Immunoblotting of the expression of stemness markers NESTIN, CD133, <t>OCT4,</t> and SOX2 in GSC2 and GSC5 cells that were treated with 1α,25(OH) 2 D 3 10 or 100 nM for 4, 8, 12, 24, and 48 h. b and c Self-renewal ability of SLCs with 1α,25(OH) 2 D 3 treatment under pH 7.4 or pH 6.8 culture conditions. Neurosphere formation assay showed the number of neurospheres (diameters larger than 50 µm) formed from GSC2 and GSC5 cells that were treated with 1α,25(OH) 2 D 3 10 or 100 nM ( b ), * P
    Anti Oct4, supplied by ABclonal, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    GP130 Overexpressing Cell Lines Show Non-Monotonic Response of Stat3 Activation to Receptor Overexpression (A) The model-predicted surface of Stat3 activation is shown as a function of time and change in production of GP130 receptors. Successive increase in GP130 production as shown by black, green, and red arrows in that order shows a non-monotonic response of Stat3 activation and the presence of a local maximum. This is illustrated by the circle in the contour map on the x–y plane. (B) GP130 overexpressing cell lines RG1–RG5 were developed to express the receptor at varying levels compared with R1 cells, and these cell lines retain Oct4 expression in the presence of LIF (C). (D) Model-predicted trends of GP130 overexpression corresponding to (A) shows normal profile of Stat3 activation (black line), Stat3 activation in slight GP130 overexpression (green line), and significant GP130 overexpression (red line). (E) Experimental results show a consistent trend in LIF-induced Stat3 activation profile using RG1 and RG5 cell lines in comparison with model results in (D). (F) IL-6 stimulation of GP130 overexpressing cell lines shows a dose-dependent increase in Stat3 activation as a function of GP130 overexpression.

    Journal: PLoS Computational Biology

    Article Title: Sensitivity Analysis of Intracellular Signaling Pathway Kinetics Predicts Targets for Stem Cell Fate Control

    doi: 10.1371/journal.pcbi.0030130

    Figure Lengend Snippet: GP130 Overexpressing Cell Lines Show Non-Monotonic Response of Stat3 Activation to Receptor Overexpression (A) The model-predicted surface of Stat3 activation is shown as a function of time and change in production of GP130 receptors. Successive increase in GP130 production as shown by black, green, and red arrows in that order shows a non-monotonic response of Stat3 activation and the presence of a local maximum. This is illustrated by the circle in the contour map on the x–y plane. (B) GP130 overexpressing cell lines RG1–RG5 were developed to express the receptor at varying levels compared with R1 cells, and these cell lines retain Oct4 expression in the presence of LIF (C). (D) Model-predicted trends of GP130 overexpression corresponding to (A) shows normal profile of Stat3 activation (black line), Stat3 activation in slight GP130 overexpression (green line), and significant GP130 overexpression (red line). (E) Experimental results show a consistent trend in LIF-induced Stat3 activation profile using RG1 and RG5 cell lines in comparison with model results in (D). (F) IL-6 stimulation of GP130 overexpressing cell lines shows a dose-dependent increase in Stat3 activation as a function of GP130 overexpression.

    Article Snippet: The following primary antibodies were used with 24-h incubation: anti-phospho-Stat3 (tyr705) (9131; Cell Signaling Technology, http://www.cellsignal.com/ ), anti-Oct3 (Oct4) (611203; BD Bioscience, http://www.bdbiosciences.com/ ), anti-phospho-Jak2 (Tyr1007–1008) (3771; Cell Signaling technology), anti-LIFR (Sc659-Santa Cruz Biotechnology, http://www.scbt.com/ ), anti- GP130 (Sc656-Santa Cruz Biotechnology), and anti-SOCS3 (Sc9023-Santa Cruz Biotechnology).

    Techniques: Activation Assay, Over Expression, Expressing

    Experimental Validation of Model Output (A) Representative images of mouse ECS for quantitative single cell fluorescence microscopy. Hoechst nuclear stain is used to determine location of nuclei, a nuclear mask is drawn, and average fluorescence levels in the mask are used to determined levels of phosphorylated Stat3 and Oct4. (B–D) Show agreement between model-predicted trends and experimental results of activation profiles of Jak phosphorylation, nuclear retention of Stat3, and SOCS3 induction, respectively. For Jak phosphorylation, nuclear accumulation of Stat3, and SOCS3 induction, both experimental and simulation results were normalized based on the maximum of the activation profiles for each of these proteins. For nuclear accumulation of phosphorylated Stat3 the results were normalized based on a steady state level in 500 pM LIF. (E) The model-predicted activation profile of nuclear retention of Tyr-705 phosphorylated Stat3 is shown in solid line and is in agreement with experimental data points.

    Journal: PLoS Computational Biology

    Article Title: Sensitivity Analysis of Intracellular Signaling Pathway Kinetics Predicts Targets for Stem Cell Fate Control

    doi: 10.1371/journal.pcbi.0030130

    Figure Lengend Snippet: Experimental Validation of Model Output (A) Representative images of mouse ECS for quantitative single cell fluorescence microscopy. Hoechst nuclear stain is used to determine location of nuclei, a nuclear mask is drawn, and average fluorescence levels in the mask are used to determined levels of phosphorylated Stat3 and Oct4. (B–D) Show agreement between model-predicted trends and experimental results of activation profiles of Jak phosphorylation, nuclear retention of Stat3, and SOCS3 induction, respectively. For Jak phosphorylation, nuclear accumulation of Stat3, and SOCS3 induction, both experimental and simulation results were normalized based on the maximum of the activation profiles for each of these proteins. For nuclear accumulation of phosphorylated Stat3 the results were normalized based on a steady state level in 500 pM LIF. (E) The model-predicted activation profile of nuclear retention of Tyr-705 phosphorylated Stat3 is shown in solid line and is in agreement with experimental data points.

    Article Snippet: The following primary antibodies were used with 24-h incubation: anti-phospho-Stat3 (tyr705) (9131; Cell Signaling Technology, http://www.cellsignal.com/ ), anti-Oct3 (Oct4) (611203; BD Bioscience, http://www.bdbiosciences.com/ ), anti-phospho-Jak2 (Tyr1007–1008) (3771; Cell Signaling technology), anti-LIFR (Sc659-Santa Cruz Biotechnology, http://www.scbt.com/ ), anti- GP130 (Sc656-Santa Cruz Biotechnology), and anti-SOCS3 (Sc9023-Santa Cruz Biotechnology).

    Techniques: Quantitative Single Cell, Fluorescence, Microscopy, Staining, Activation Assay

    Desensitization of ESCs to LIF Stimulation Can Be Investigated Computationally and Used to Control Self-Renewal (A) Model-predicted trends of desensitization of Stat3 activation to LIF stimulation. Color bar inset shows history of 500 pM LIF stimulations. (B) Experimental results are in agreement with model-predicted desensitization kinetics in (A) and show a gradual loss of desensitization in absence of ligand. (C) Global sensitivity analysis shows parameter interactions which result in right-shift (positive minutes) or left-shift (negative minutes) in restimulation profile of Stat3. Shift in the profile was determined from the maximum of cross-correlation of two consecutive Stat3 activation profiles. (D) Sensitivity analysis clustergram shows Euclidean distance measurement between two consecutive Stat3 activation profiles and distinguishes parameter interactions which influence desensitization by changing the restimulation profile. (E) Experimental results of dose response of Stat3 activation to LIF show the steady state response as well as the peak values of Stat3 activation during transient LIF stimulation. (F) Dose response of Oct4 expression after 72 h at different LIF concentrations. (G) Stat3 activation profiles for 10 pM LIF stimulation with 1-h and 6-h periods in red and blue, respectively. During 6-h cycles, LIF is removed for 3 h during which time a lower Stat3 activation level is observed in comparison with the 1-h period. (H) Histograms of Oct4 expression show that 1-hr period of LIF stimulation (red) maintains a higher percentage of Oct4+ cells than 6-h period of LIF stimulation after 72 h. (I) Oct4 expression of cells maintained for 72 h in different conditions shows that the percentage of Oct4+ cells in the 1-h period is higher than the 6-h period and comparable to 500 pM constant LIF levels.

    Journal: PLoS Computational Biology

    Article Title: Sensitivity Analysis of Intracellular Signaling Pathway Kinetics Predicts Targets for Stem Cell Fate Control

    doi: 10.1371/journal.pcbi.0030130

    Figure Lengend Snippet: Desensitization of ESCs to LIF Stimulation Can Be Investigated Computationally and Used to Control Self-Renewal (A) Model-predicted trends of desensitization of Stat3 activation to LIF stimulation. Color bar inset shows history of 500 pM LIF stimulations. (B) Experimental results are in agreement with model-predicted desensitization kinetics in (A) and show a gradual loss of desensitization in absence of ligand. (C) Global sensitivity analysis shows parameter interactions which result in right-shift (positive minutes) or left-shift (negative minutes) in restimulation profile of Stat3. Shift in the profile was determined from the maximum of cross-correlation of two consecutive Stat3 activation profiles. (D) Sensitivity analysis clustergram shows Euclidean distance measurement between two consecutive Stat3 activation profiles and distinguishes parameter interactions which influence desensitization by changing the restimulation profile. (E) Experimental results of dose response of Stat3 activation to LIF show the steady state response as well as the peak values of Stat3 activation during transient LIF stimulation. (F) Dose response of Oct4 expression after 72 h at different LIF concentrations. (G) Stat3 activation profiles for 10 pM LIF stimulation with 1-h and 6-h periods in red and blue, respectively. During 6-h cycles, LIF is removed for 3 h during which time a lower Stat3 activation level is observed in comparison with the 1-h period. (H) Histograms of Oct4 expression show that 1-hr period of LIF stimulation (red) maintains a higher percentage of Oct4+ cells than 6-h period of LIF stimulation after 72 h. (I) Oct4 expression of cells maintained for 72 h in different conditions shows that the percentage of Oct4+ cells in the 1-h period is higher than the 6-h period and comparable to 500 pM constant LIF levels.

    Article Snippet: The following primary antibodies were used with 24-h incubation: anti-phospho-Stat3 (tyr705) (9131; Cell Signaling Technology, http://www.cellsignal.com/ ), anti-Oct3 (Oct4) (611203; BD Bioscience, http://www.bdbiosciences.com/ ), anti-phospho-Jak2 (Tyr1007–1008) (3771; Cell Signaling technology), anti-LIFR (Sc659-Santa Cruz Biotechnology, http://www.scbt.com/ ), anti- GP130 (Sc656-Santa Cruz Biotechnology), and anti-SOCS3 (Sc9023-Santa Cruz Biotechnology).

    Techniques: Activation Assay, Expressing

    Hes1 regulates expression of Notch signaling and cell cycle factors during differentiation. (A) mRNA levels of Notch signaling genes, Dll1 , Jag1, Hes5 and Notch1 . (B) Protein levels of Notch1 receptor, Notch intracellular domain (NICD), p57, Oct3/4 and Hes1 in the wild-type cells (WT; lanes 1, 4, 7 and 10) and in Hes1-sustained cells (R5; lanes 2, 5, 8 and 11, R6; lanes 3, 6, 9 and 12) under a neural differentiation condition.

    Journal: Genes to Cells

    Article Title: Hes1 regulates embryonic stem cell differentiation by suppressing Notch signaling

    doi: 10.1111/j.1365-2443.2010.01413.x

    Figure Lengend Snippet: Hes1 regulates expression of Notch signaling and cell cycle factors during differentiation. (A) mRNA levels of Notch signaling genes, Dll1 , Jag1, Hes5 and Notch1 . (B) Protein levels of Notch1 receptor, Notch intracellular domain (NICD), p57, Oct3/4 and Hes1 in the wild-type cells (WT; lanes 1, 4, 7 and 10) and in Hes1-sustained cells (R5; lanes 2, 5, 8 and 11, R6; lanes 3, 6, 9 and 12) under a neural differentiation condition.

    Article Snippet: For immunocytochemistry, the following antibodies were used: rabbit anti-Tuj-1 antibody (Covance), mouse anti-Nestin, anti-Oct3/4 antibodies (BD Pharmingen) and rabbit anti-Hes1 antibody ( ).

    Techniques: Expressing

    Hes1 knockdown promotes further mesodermal differentiation in Hes1-sustained cells. mRNA kinetics of marker genes, Oct3/4 , Gata4 and Brachyury (A), neural marker genes, Mash1 , Sox1 and Nestin (B), and mesodermal lineage marker genes, Nkx-2.5 and Flk-1 (C) were measured by quantitative real-time PCR. Arrows show the infection of lentivirus vectors carrying shRNA of scramble (blue) or Hes1 knockdown (Hes1-KD, red) into Hes1-sustained cells on day 6. Cells without virus infection were also analyzed (green).

    Journal: Genes to Cells

    Article Title: Hes1 regulates embryonic stem cell differentiation by suppressing Notch signaling

    doi: 10.1111/j.1365-2443.2010.01413.x

    Figure Lengend Snippet: Hes1 knockdown promotes further mesodermal differentiation in Hes1-sustained cells. mRNA kinetics of marker genes, Oct3/4 , Gata4 and Brachyury (A), neural marker genes, Mash1 , Sox1 and Nestin (B), and mesodermal lineage marker genes, Nkx-2.5 and Flk-1 (C) were measured by quantitative real-time PCR. Arrows show the infection of lentivirus vectors carrying shRNA of scramble (blue) or Hes1 knockdown (Hes1-KD, red) into Hes1-sustained cells on day 6. Cells without virus infection were also analyzed (green).

    Article Snippet: For immunocytochemistry, the following antibodies were used: rabbit anti-Tuj-1 antibody (Covance), mouse anti-Nestin, anti-Oct3/4 antibodies (BD Pharmingen) and rabbit anti-Hes1 antibody ( ).

    Techniques: Marker, Real-time Polymerase Chain Reaction, Infection, shRNA

    Hes1 protein expression in Hes1-sustained embryonic stem (ES) cells. (A) The structure for sustained Hes1 expression. The Hes1 cDNA with the IRES-EGFP sequence was knocked-in into the Rosa26 locus, so that Hes1 and EGFP were constitutively expressed from the Rosa26 promoter ( Kobayashi et al. 2009 ). (B) Hes1 and Oct3/4 expression in the wild-type (WT) and Hes1-sustained (R5, R6) ES cell lines. Actin is a loading control. (C) Immunostaining of Hes1 (red) with DAPI staining (blue) in ES cell lines.

    Journal: Genes to Cells

    Article Title: Hes1 regulates embryonic stem cell differentiation by suppressing Notch signaling

    doi: 10.1111/j.1365-2443.2010.01413.x

    Figure Lengend Snippet: Hes1 protein expression in Hes1-sustained embryonic stem (ES) cells. (A) The structure for sustained Hes1 expression. The Hes1 cDNA with the IRES-EGFP sequence was knocked-in into the Rosa26 locus, so that Hes1 and EGFP were constitutively expressed from the Rosa26 promoter ( Kobayashi et al. 2009 ). (B) Hes1 and Oct3/4 expression in the wild-type (WT) and Hes1-sustained (R5, R6) ES cell lines. Actin is a loading control. (C) Immunostaining of Hes1 (red) with DAPI staining (blue) in ES cell lines.

    Article Snippet: For immunocytochemistry, the following antibodies were used: rabbit anti-Tuj-1 antibody (Covance), mouse anti-Nestin, anti-Oct3/4 antibodies (BD Pharmingen) and rabbit anti-Hes1 antibody ( ).

    Techniques: Expressing, Sequencing, Immunostaining, Staining

    Kinetics of marker gene expression under a neural differentiation condition. mRNA levels of marker genes in the control (WT; blue) and Hes1-sustained embryonic stem cells (R5 and R6; red) under a neural differentiation condition were analyzed by quantitative real-time PCR. Each value was given in the ratio to the wild-type cells (WT) on day 0. (A) mRNA levels of marker genes, Oct3/4 , Fgf5 , Brachyury and Gata4. (B) mRNA levels of neural marker genes, Mash1 , Nestin and Tuj-1 .

    Journal: Genes to Cells

    Article Title: Hes1 regulates embryonic stem cell differentiation by suppressing Notch signaling

    doi: 10.1111/j.1365-2443.2010.01413.x

    Figure Lengend Snippet: Kinetics of marker gene expression under a neural differentiation condition. mRNA levels of marker genes in the control (WT; blue) and Hes1-sustained embryonic stem cells (R5 and R6; red) under a neural differentiation condition were analyzed by quantitative real-time PCR. Each value was given in the ratio to the wild-type cells (WT) on day 0. (A) mRNA levels of marker genes, Oct3/4 , Fgf5 , Brachyury and Gata4. (B) mRNA levels of neural marker genes, Mash1 , Nestin and Tuj-1 .

    Article Snippet: For immunocytochemistry, the following antibodies were used: rabbit anti-Tuj-1 antibody (Covance), mouse anti-Nestin, anti-Oct3/4 antibodies (BD Pharmingen) and rabbit anti-Hes1 antibody ( ).

    Techniques: Marker, Expressing, Real-time Polymerase Chain Reaction

    Comparison of marker protein expression under a neural differentiation condition. The control (WT, upper panels) and Hes1-sustained cells (R6, lower panels) cultured in N2B27 medium were analyzed on days 4 and 6 by immunocytochemistry using anti-Oct3/4, anti-Nestin and anti-Tuj-1 antibodies (red) with DAPI staining (blue). Scale bars, 100 μm.

    Journal: Genes to Cells

    Article Title: Hes1 regulates embryonic stem cell differentiation by suppressing Notch signaling

    doi: 10.1111/j.1365-2443.2010.01413.x

    Figure Lengend Snippet: Comparison of marker protein expression under a neural differentiation condition. The control (WT, upper panels) and Hes1-sustained cells (R6, lower panels) cultured in N2B27 medium were analyzed on days 4 and 6 by immunocytochemistry using anti-Oct3/4, anti-Nestin and anti-Tuj-1 antibodies (red) with DAPI staining (blue). Scale bars, 100 μm.

    Article Snippet: For immunocytochemistry, the following antibodies were used: rabbit anti-Tuj-1 antibody (Covance), mouse anti-Nestin, anti-Oct3/4 antibodies (BD Pharmingen) and rabbit anti-Hes1 antibody ( ).

    Techniques: Marker, Expressing, Cell Culture, Immunocytochemistry, Staining

    Contribution of rat EG cells to embryonic chimaeras including germ cells. ( A ) Phase contrast and fluorescence images of Red1.2 EG cell clone. ( B ) Graph showing chromosome number per cell in 50 metaphase spreads from line Red1.2. Inset is a karyotype of Red1.2, showing 42 chromosomes. ( C ) Bright-field and fluorescence images of E13.5 chimaeric embryos. ( D ) Confocal section of genital ridge and surrounding mesonephric tissue dissected from a chimaeric embryo and immunostained for DsRed and Oct4. ( E ) Higher magnification confocal section of D shows cells co-stained for DsRed and Oct4, demonstrating the contribution of EG cells to germ cells. Scale bars: 100 μm in A,D; 25 μm in E.

    Journal: Development (Cambridge, England)

    Article Title: Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state

    doi: 10.1242/dev.050427

    Figure Lengend Snippet: Contribution of rat EG cells to embryonic chimaeras including germ cells. ( A ) Phase contrast and fluorescence images of Red1.2 EG cell clone. ( B ) Graph showing chromosome number per cell in 50 metaphase spreads from line Red1.2. Inset is a karyotype of Red1.2, showing 42 chromosomes. ( C ) Bright-field and fluorescence images of E13.5 chimaeric embryos. ( D ) Confocal section of genital ridge and surrounding mesonephric tissue dissected from a chimaeric embryo and immunostained for DsRed and Oct4. ( E ) Higher magnification confocal section of D shows cells co-stained for DsRed and Oct4, demonstrating the contribution of EG cells to germ cells. Scale bars: 100 μm in A,D; 25 μm in E.

    Article Snippet: Primary antibodies were: Nanog (Abcam, 1:200; Cosmo Bio, 1:500), Oct4 (BD, 1:200; Santa Cruz, 1:200), Sox2 (Abcam, 1:100), βIII tubulin (Sigma, 1:200), myosin (MF-20, 1:5) and DsRed (Clontech, 1:1000).

    Techniques: Fluorescence, Staining

    Mouse EG cells can be maintained and derived in 2i-LIF. ( A ) Phase contrast and fluorescence images of Oct4-ΔPE-GFP EG cells. ( B ) Phase contrast and fluorescence images of Oct4-ΔPE-GFP EG cells cultured in: FCS-LIF on a MEF feeder layer; FCS-LIF after one passage without feeders; 2i-LIF without feeders. ( C ) Flow cytometry analysis of Oct4-ΔPE-GFP EG cells cultured in 2i-LIF compared with FCS-LIF on feeders. Gates were set to eliminate feeders and cell debris. ( D ) Colony-forming assay on Oct4-ΔPE-GFP EG cells. Colonies were scored for alkaline phosphatase (AP) and designated positive, negative or mixed. Data are means of two biological replicates. ( E ) Schematic of EG cell derivation protocol. On day 3, medium was changed to either FCS-LIF or 2i-LIF. ( F ) Comparison of GFP-positive EG cell colony formation following addition of 2i-LIF on day 3 compared with FCS-LIF. ( G ) High-contribution coat colour chimaeras generated with agouti EG cells derived in 2i-LIF injected into C57BL/6 blastocysts. ( H ) Chimaera, C57BL/6 mate, and mixed litter of agouti and black pups. Scale bars: 100 μm in A; 25 μm in B.

    Journal: Development (Cambridge, England)

    Article Title: Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state

    doi: 10.1242/dev.050427

    Figure Lengend Snippet: Mouse EG cells can be maintained and derived in 2i-LIF. ( A ) Phase contrast and fluorescence images of Oct4-ΔPE-GFP EG cells. ( B ) Phase contrast and fluorescence images of Oct4-ΔPE-GFP EG cells cultured in: FCS-LIF on a MEF feeder layer; FCS-LIF after one passage without feeders; 2i-LIF without feeders. ( C ) Flow cytometry analysis of Oct4-ΔPE-GFP EG cells cultured in 2i-LIF compared with FCS-LIF on feeders. Gates were set to eliminate feeders and cell debris. ( D ) Colony-forming assay on Oct4-ΔPE-GFP EG cells. Colonies were scored for alkaline phosphatase (AP) and designated positive, negative or mixed. Data are means of two biological replicates. ( E ) Schematic of EG cell derivation protocol. On day 3, medium was changed to either FCS-LIF or 2i-LIF. ( F ) Comparison of GFP-positive EG cell colony formation following addition of 2i-LIF on day 3 compared with FCS-LIF. ( G ) High-contribution coat colour chimaeras generated with agouti EG cells derived in 2i-LIF injected into C57BL/6 blastocysts. ( H ) Chimaera, C57BL/6 mate, and mixed litter of agouti and black pups. Scale bars: 100 μm in A; 25 μm in B.

    Article Snippet: Primary antibodies were: Nanog (Abcam, 1:200; Cosmo Bio, 1:500), Oct4 (BD, 1:200; Santa Cruz, 1:200), Sox2 (Abcam, 1:100), βIII tubulin (Sigma, 1:200), myosin (MF-20, 1:5) and DsRed (Clontech, 1:1000).

    Techniques: Derivative Assay, Fluorescence, Cell Culture, Flow Cytometry, Cytometry, Generated, Injection

    Pre-migratory rat PGCs co-express Oct4 and Blimp1. ( A ) Bright-field image of an E10 rat embryo. Dashed line depicts line of incision to isolate the posterior fragment. ( B ) Bright-field image of an isolated posterior fragment of E10 rat embryo. ( C ) Confocal section through the posterior fragment of an E10 rat embryo immunostained in wholemount for Oct4 (green) and co-stained with DAPI (blue outline). ( D ) Higher magnification confocal section through a posterior embryo fragment showing nuclear staining of Oct4 in a cluster of cells at the base of the allantois. ( E ) Double immunostaining for Oct4 and Blimp1 on a cryosection through the posterior fragment. Scale bars: 100 μm in C, 10 μm in D,E.

    Journal: Development (Cambridge, England)

    Article Title: Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state

    doi: 10.1242/dev.050427

    Figure Lengend Snippet: Pre-migratory rat PGCs co-express Oct4 and Blimp1. ( A ) Bright-field image of an E10 rat embryo. Dashed line depicts line of incision to isolate the posterior fragment. ( B ) Bright-field image of an isolated posterior fragment of E10 rat embryo. ( C ) Confocal section through the posterior fragment of an E10 rat embryo immunostained in wholemount for Oct4 (green) and co-stained with DAPI (blue outline). ( D ) Higher magnification confocal section through a posterior embryo fragment showing nuclear staining of Oct4 in a cluster of cells at the base of the allantois. ( E ) Double immunostaining for Oct4 and Blimp1 on a cryosection through the posterior fragment. Scale bars: 100 μm in C, 10 μm in D,E.

    Article Snippet: Primary antibodies were: Nanog (Abcam, 1:200; Cosmo Bio, 1:500), Oct4 (BD, 1:200; Santa Cruz, 1:200), Sox2 (Abcam, 1:100), βIII tubulin (Sigma, 1:200), myosin (MF-20, 1:5) and DsRed (Clontech, 1:1000).

    Techniques: Isolation, Staining, Double Immunostaining

    Rat EG cells can be derived and expanded in 2i-LIF and express markers of pluripotency. ( A ) Phase contrast image of a primary rat EG cell colony. ( B , C ) Phase contrast images of rat EG cells after 5 (B) and 35 (C) passages. ( D ) Oct4, Sox2 and Nanog immunostaining of rat EG cells. ( E ) Alkaline phosphatase (AP) staining of rat EG cells. ( F ) RT-PCR analysis of pluripotency markers in three early-passage rat EG cell lines (WBY1 P7, WBY2 P7, WBY3 P6) and two late-passage rat EG cell lines (WBY2 P35, WBY7 P33). Controls are rat ES cells (DAX11), rat neural stem (rNS) cells (Ras2-GFP), posterior fragments of E10 embryos and whole E13.5 genital ridges. ( G ) Colony-forming assay on dissociated rat EG cells plated on laminin. Colonies were scored for AP and designated positive or negative. Data are means of two biological replicates. ( H ) Typical AP-positive colony. Scale bars: 100 μm in A-C,E; 25 μm in D.

    Journal: Development (Cambridge, England)

    Article Title: Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state

    doi: 10.1242/dev.050427

    Figure Lengend Snippet: Rat EG cells can be derived and expanded in 2i-LIF and express markers of pluripotency. ( A ) Phase contrast image of a primary rat EG cell colony. ( B , C ) Phase contrast images of rat EG cells after 5 (B) and 35 (C) passages. ( D ) Oct4, Sox2 and Nanog immunostaining of rat EG cells. ( E ) Alkaline phosphatase (AP) staining of rat EG cells. ( F ) RT-PCR analysis of pluripotency markers in three early-passage rat EG cell lines (WBY1 P7, WBY2 P7, WBY3 P6) and two late-passage rat EG cell lines (WBY2 P35, WBY7 P33). Controls are rat ES cells (DAX11), rat neural stem (rNS) cells (Ras2-GFP), posterior fragments of E10 embryos and whole E13.5 genital ridges. ( G ) Colony-forming assay on dissociated rat EG cells plated on laminin. Colonies were scored for AP and designated positive or negative. Data are means of two biological replicates. ( H ) Typical AP-positive colony. Scale bars: 100 μm in A-C,E; 25 μm in D.

    Article Snippet: Primary antibodies were: Nanog (Abcam, 1:200; Cosmo Bio, 1:500), Oct4 (BD, 1:200; Santa Cruz, 1:200), Sox2 (Abcam, 1:100), βIII tubulin (Sigma, 1:200), myosin (MF-20, 1:5) and DsRed (Clontech, 1:1000).

    Techniques: Derivative Assay, Immunostaining, Staining, Reverse Transcription Polymerase Chain Reaction

    SAHA induces differentiation of enriched CSCs in NE1.8 cells. ( a ) Effects of SAHA treatment on cell populations of CD44 + /CD24 −/low , Oct3/4, and CD133 in NE1.8 cells; top: representative results of flow cytometric analysis; bottom: diagram showing the percentages of cell populations with positive staining of indicated marker. ( b ) Effect of SAHA treatment on Sox2 expression in NE1.8 cells; top: representative results of flow cytometric analysis; bottom: diagram showing the percentages of the cell subpopulation with weak expression of Sox2 as circulated. ( c ) Effect of SAHA treatment on NSE expression in NE1.8 cells; top: representative results of flow cytometric analysis; bottom: diagram showing the percentages of cell populations with negative NSE staining as circulated. ( d ) Western blots showing that SAHA treatment induces expression of differentiation markers of involucrin and Syndecan 3 proteins in NE1.8 cells. β-actin was included for equivalent protein loading. ( e ) Images of collected tumors from tumor initiating test with SAHA-treated NE1.8 cells. ( f ) HE and IHC staining of NSE in a tumor specimen collected from the tumor initiating test with SAHA-treated NE1.8 cells. ( g ) Western blots showing that the effects of SAHA treatment on activation and expressions of Akt, and on AUKRA protein level in NE1.8 cells. P values were determined from at least three independent experiments. Error bars indicate standard deviation. SAHA: suberoylanilide hydroxamic acid; CSC: cancer stem cell; NSE: neuron-specific enolase; HE: hematoxylin and eosin stain; IHC: immunohistochemistry; FITC: fluorescein isothiocyanate.

    Journal: Asian Journal of Andrology

    Article Title: Potential therapeutic effect of epigenetic therapy on treatment-induced neuroendocrine prostate cancer

    doi: 10.4103/1008-682X.191518

    Figure Lengend Snippet: SAHA induces differentiation of enriched CSCs in NE1.8 cells. ( a ) Effects of SAHA treatment on cell populations of CD44 + /CD24 −/low , Oct3/4, and CD133 in NE1.8 cells; top: representative results of flow cytometric analysis; bottom: diagram showing the percentages of cell populations with positive staining of indicated marker. ( b ) Effect of SAHA treatment on Sox2 expression in NE1.8 cells; top: representative results of flow cytometric analysis; bottom: diagram showing the percentages of the cell subpopulation with weak expression of Sox2 as circulated. ( c ) Effect of SAHA treatment on NSE expression in NE1.8 cells; top: representative results of flow cytometric analysis; bottom: diagram showing the percentages of cell populations with negative NSE staining as circulated. ( d ) Western blots showing that SAHA treatment induces expression of differentiation markers of involucrin and Syndecan 3 proteins in NE1.8 cells. β-actin was included for equivalent protein loading. ( e ) Images of collected tumors from tumor initiating test with SAHA-treated NE1.8 cells. ( f ) HE and IHC staining of NSE in a tumor specimen collected from the tumor initiating test with SAHA-treated NE1.8 cells. ( g ) Western blots showing that the effects of SAHA treatment on activation and expressions of Akt, and on AUKRA protein level in NE1.8 cells. P values were determined from at least three independent experiments. Error bars indicate standard deviation. SAHA: suberoylanilide hydroxamic acid; CSC: cancer stem cell; NSE: neuron-specific enolase; HE: hematoxylin and eosin stain; IHC: immunohistochemistry; FITC: fluorescein isothiocyanate.

    Article Snippet: Cells were then stained with PE-conjugated anti-Sox2, anti-Oct3/4, and anti-Nanog antibodies (BD Biosciences, San Jose, CA, USA), or stained with PE-conjugated anti-CD133 (Miltenyi Biotec, San Diego, CA, USA), or co-stained with PE-conjugated anti-CD24 (BD Biosciences, San Jose, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated anti-CD44 antibodies (BD Biosciences, San Jose, CA, USA).

    Techniques: Flow Cytometry, Staining, Marker, Expressing, Western Blot, Immunohistochemistry, Activation Assay, Standard Deviation, H&E Stain

    Flow cytometric analysis for stem cell surface markers. Increased cell fractions with positive staining of putative stem cell markers CD133 ( a ), Oct3/4 ( b ), and CD44 + /CD24 −/low ( c ) in NE1.8 cells compared to parental LNCaP cells. Top: flow cytometric analysis; bottom: diagram showing the percentages of cell populations with positive staining. P values were determined from at least three independent experiments. Error bars indicate standard deviation. ( d ) Flow cytometric analysis for putative stem-cell surface markers Sox2 (left) and Nanog (right) in NE1.8 and LNCaP cells. ( e ) Flow cytometric analysis showing the shift of cell population toward positive staining for Sox2 in NE1.8 cells when compared to LNCaP cells. FITC: fluorescein isothiocyanate.

    Journal: Asian Journal of Andrology

    Article Title: Potential therapeutic effect of epigenetic therapy on treatment-induced neuroendocrine prostate cancer

    doi: 10.4103/1008-682X.191518

    Figure Lengend Snippet: Flow cytometric analysis for stem cell surface markers. Increased cell fractions with positive staining of putative stem cell markers CD133 ( a ), Oct3/4 ( b ), and CD44 + /CD24 −/low ( c ) in NE1.8 cells compared to parental LNCaP cells. Top: flow cytometric analysis; bottom: diagram showing the percentages of cell populations with positive staining. P values were determined from at least three independent experiments. Error bars indicate standard deviation. ( d ) Flow cytometric analysis for putative stem-cell surface markers Sox2 (left) and Nanog (right) in NE1.8 and LNCaP cells. ( e ) Flow cytometric analysis showing the shift of cell population toward positive staining for Sox2 in NE1.8 cells when compared to LNCaP cells. FITC: fluorescein isothiocyanate.

    Article Snippet: Cells were then stained with PE-conjugated anti-Sox2, anti-Oct3/4, and anti-Nanog antibodies (BD Biosciences, San Jose, CA, USA), or stained with PE-conjugated anti-CD133 (Miltenyi Biotec, San Diego, CA, USA), or co-stained with PE-conjugated anti-CD24 (BD Biosciences, San Jose, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated anti-CD44 antibodies (BD Biosciences, San Jose, CA, USA).

    Techniques: Flow Cytometry, Staining, Standard Deviation

    Effects of ADT treatment on CSC enrichment in LNCaP cells. ( a ) Effects of ADT treatment on expression of putative stem-cell surface markers of Oct3/4 and CD133 in LNCaP cells; top: representative results of flow cytometric analysis; bottom: diagram showing the percentages of cell populations with positive staining for indicated marker. ( b ) Effects of ADT treatment on CD44 expression in LNCaP cells; top left: flow cytometric analysis of gating control for LNCaP cells without ADT treatment; top right: flow cytometric analysis of CD44 for LNCaP cells without ADT treatment; middle left: flow cytometric analysis of gating control for LNCaP cells with ADT treatment; middle right: flow cytometric analysis of CD44 for LNCaP cells with ADT treatment; bottom: diagram showing the percentages of cell populations with positive CD44 staining. P values were determined from at least three independent experiments. Error bars indicate standard deviation. ADT: androgen deprivation treatment; CSC: cancer stem cell; FITC: fluorescein isothiocyanate.

    Journal: Asian Journal of Andrology

    Article Title: Potential therapeutic effect of epigenetic therapy on treatment-induced neuroendocrine prostate cancer

    doi: 10.4103/1008-682X.191518

    Figure Lengend Snippet: Effects of ADT treatment on CSC enrichment in LNCaP cells. ( a ) Effects of ADT treatment on expression of putative stem-cell surface markers of Oct3/4 and CD133 in LNCaP cells; top: representative results of flow cytometric analysis; bottom: diagram showing the percentages of cell populations with positive staining for indicated marker. ( b ) Effects of ADT treatment on CD44 expression in LNCaP cells; top left: flow cytometric analysis of gating control for LNCaP cells without ADT treatment; top right: flow cytometric analysis of CD44 for LNCaP cells without ADT treatment; middle left: flow cytometric analysis of gating control for LNCaP cells with ADT treatment; middle right: flow cytometric analysis of CD44 for LNCaP cells with ADT treatment; bottom: diagram showing the percentages of cell populations with positive CD44 staining. P values were determined from at least three independent experiments. Error bars indicate standard deviation. ADT: androgen deprivation treatment; CSC: cancer stem cell; FITC: fluorescein isothiocyanate.

    Article Snippet: Cells were then stained with PE-conjugated anti-Sox2, anti-Oct3/4, and anti-Nanog antibodies (BD Biosciences, San Jose, CA, USA), or stained with PE-conjugated anti-CD133 (Miltenyi Biotec, San Diego, CA, USA), or co-stained with PE-conjugated anti-CD24 (BD Biosciences, San Jose, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated anti-CD44 antibodies (BD Biosciences, San Jose, CA, USA).

    Techniques: Expressing, Flow Cytometry, Staining, Marker, Standard Deviation

    Activation of Wnt signaling increased ß-catenin signals and Wnt target gene expressions. ( a – c ) Immunohistochemistry was performed on control and CHIR-treated (for 48 h) ESC colonies using ß-catenin with phalloidin ( a ), GFP ( b ), Nanog and Oct3/4 ( c ) antibodies. Scale bars 25 μm. ( d ) FACS analysis of ß-catenin-mEGFP line. ( e ) Quantification of ß-catenin-mEGFP positive cells by FACS. ( f ) Quantification of axin2 and dkk1 expression via RT-qPCR, following CHIR treatment for 48 h. Error bars indicate standard error of the mean of each experiment. ( g ) Merged images of transillumination (Trans) and 7Tcf::Cherry signals of ESC colonies by addition of 10 μM CHIR for 48 h. Scale bar 100 μm. Transillumination images of single ESC colony in control, CHIR- (10 μM) and Wnt3a- (200 ng/ml) treated condition (for 48 h).

    Journal: In Vitro Cellular & Developmental Biology. Animal

    Article Title: Activation of Wnt/ß-catenin signaling in ESC promotes rostral forebrain differentiation in vitro

    doi: 10.1007/s11626-015-9975-y

    Figure Lengend Snippet: Activation of Wnt signaling increased ß-catenin signals and Wnt target gene expressions. ( a – c ) Immunohistochemistry was performed on control and CHIR-treated (for 48 h) ESC colonies using ß-catenin with phalloidin ( a ), GFP ( b ), Nanog and Oct3/4 ( c ) antibodies. Scale bars 25 μm. ( d ) FACS analysis of ß-catenin-mEGFP line. ( e ) Quantification of ß-catenin-mEGFP positive cells by FACS. ( f ) Quantification of axin2 and dkk1 expression via RT-qPCR, following CHIR treatment for 48 h. Error bars indicate standard error of the mean of each experiment. ( g ) Merged images of transillumination (Trans) and 7Tcf::Cherry signals of ESC colonies by addition of 10 μM CHIR for 48 h. Scale bar 100 μm. Transillumination images of single ESC colony in control, CHIR- (10 μM) and Wnt3a- (200 ng/ml) treated condition (for 48 h).

    Article Snippet: After incubation of ESCs in 2% skim milk/PBS blocking solution, we used specific antibodies in blocking solution as follows: ß-catenin (mouse, 1:500, BD transduction 610153: rabbit, 1:500, C-2206; Sigma-aldrich): GFP (rat, 1/500, 04404–84; Nacalai, San Diego, CA): Nanog (rabbit, 1/1000, RCAB0001P; ReproCell, Yokohama, Japan): Oct3/4 (mouse, 1/200, BD 611202).

    Techniques: Activation Assay, Immunohistochemistry, FACS, Expressing, Quantitative RT-PCR

    FGF4 and heparin rescue neural differentiation of NDST1 −/− NDST2 −/− ES cells. A , the addition of conditioned medium from WT ES cells subjected to neural differentiation to the NDST1 −/− NDST2 −/− cells rescued neural differentiation of the mutant cells ( top , day 13). The addition of 10 ng/ml FGF4 only for 7 days had no effect on mutant cells in N2B27 ( middle ), whereas 10 ng/ml FGF4 in combination with 0.1 μg/ml heparin rescued neural differentiation ( bottom , day 9). B , immunofluorescence staining of mutant cells. Shown is neural differentiation in the presence of FGF4 and heparin ( top ) and staining for nestin ( red ) and DAPI ( blue ). Mutant neural cells were plated on a polyornithine/fibronectin-coated surface 5 days after withdrawal of FGF4 and heparin and stained for the neuronal marker Tuj1 ( red ), the astrocyte marker GFAP ( green ), and DAPI ( blue ). C , quantification of Oct3/4 and nestin-expressing NDST1 −/− NDST2 −/− cells subjected to 6 days of neural differentiation in N2B27 medium with and without the addition of FGF4 and heparin. Error bars , S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Undersulfation of Heparan Sulfate Restricts Differentiation Potential of Mouse Embryonic Stem Cells *

    doi: 10.1074/jbc.M111.337030

    Figure Lengend Snippet: FGF4 and heparin rescue neural differentiation of NDST1 −/− NDST2 −/− ES cells. A , the addition of conditioned medium from WT ES cells subjected to neural differentiation to the NDST1 −/− NDST2 −/− cells rescued neural differentiation of the mutant cells ( top , day 13). The addition of 10 ng/ml FGF4 only for 7 days had no effect on mutant cells in N2B27 ( middle ), whereas 10 ng/ml FGF4 in combination with 0.1 μg/ml heparin rescued neural differentiation ( bottom , day 9). B , immunofluorescence staining of mutant cells. Shown is neural differentiation in the presence of FGF4 and heparin ( top ) and staining for nestin ( red ) and DAPI ( blue ). Mutant neural cells were plated on a polyornithine/fibronectin-coated surface 5 days after withdrawal of FGF4 and heparin and stained for the neuronal marker Tuj1 ( red ), the astrocyte marker GFAP ( green ), and DAPI ( blue ). C , quantification of Oct3/4 and nestin-expressing NDST1 −/− NDST2 −/− cells subjected to 6 days of neural differentiation in N2B27 medium with and without the addition of FGF4 and heparin. Error bars , S.D.

    Article Snippet: Primary antibodies were Oct3/4 (1:100; Santa Cruz Biotechnology, Inc.), nestin (1:200; Chemicon), β-III-tubulin (Tuj1, 1:500; Covance), and glial fibrillary acidic protein (GFAP) (1:200; Sigma-Aldrich).

    Techniques: Mutagenesis, Immunofluorescence, Staining, Marker, Expressing

    NDST1 −/− NDST2 −/− ES cells are blocked in differentiation to the neural lineage. A , WT ES cells differentiate to neural cells, whereas mutant ES cells appear morphologically undifferentiated after 6 days in N2B27 medium. B , growth curve comparing WT and mutant cell numbers during 13 days of differentiation in N2B27 medium. Three different mutant ES cell lines were tested, A1, A3, and B5. C , immunofluorescent staining of WT and mutant cells for the neural precursor marker nestin ( red ) and nucleus DAPI ( blue ) after 6 days of differentiation. D , undifferentiated ES cells ( top panels ) and cells differentiated for 6 days ( bottom panels ) were stained for alkaline phosphatase ( purple , WT cells ( left panels ) and mutant ES cells ( middle panels )). Right panels , immunofluorescent staining of mutant ES cells for Oct3/4 ( red ).

    Journal: The Journal of Biological Chemistry

    Article Title: Undersulfation of Heparan Sulfate Restricts Differentiation Potential of Mouse Embryonic Stem Cells *

    doi: 10.1074/jbc.M111.337030

    Figure Lengend Snippet: NDST1 −/− NDST2 −/− ES cells are blocked in differentiation to the neural lineage. A , WT ES cells differentiate to neural cells, whereas mutant ES cells appear morphologically undifferentiated after 6 days in N2B27 medium. B , growth curve comparing WT and mutant cell numbers during 13 days of differentiation in N2B27 medium. Three different mutant ES cell lines were tested, A1, A3, and B5. C , immunofluorescent staining of WT and mutant cells for the neural precursor marker nestin ( red ) and nucleus DAPI ( blue ) after 6 days of differentiation. D , undifferentiated ES cells ( top panels ) and cells differentiated for 6 days ( bottom panels ) were stained for alkaline phosphatase ( purple , WT cells ( left panels ) and mutant ES cells ( middle panels )). Right panels , immunofluorescent staining of mutant ES cells for Oct3/4 ( red ).

    Article Snippet: Primary antibodies were Oct3/4 (1:100; Santa Cruz Biotechnology, Inc.), nestin (1:200; Chemicon), β-III-tubulin (Tuj1, 1:500; Covance), and glial fibrillary acidic protein (GFAP) (1:200; Sigma-Aldrich).

    Techniques: Mutagenesis, Staining, Marker

    Gene expression analysis of WT and NDST1 −/− NDST2 −/− ES cells, before and after 6 days of neural differentiation. WT and mutant ES cells were cultured in ES cell medium ( Undiff ) or for 6 days in N2B27 medium to promote neural differentiation ( Diff ). Hypoxanthine-guanine phosphoribosyltransferase ( HPRT ) was used as an internal control ( A , top ). A–C , semiquantitative RT-PCR analysis. A , expression of the undifferentiated markers Oct3/4 and nanog, neural precursor protein nestin, mesoderm marker Brachyury, and primitive endoderm marker GATA4. B , expression of BMP4 and the BMP inhibitors noggin, chordin, and follistatin. C , expression of FGF4, FGF5, and the FGF receptors 1b, 1c, 2b, 2c, 3, and 4. D , real-time RT-PCR analysis of NDST1, -2, -3, and -4 transcripts. For each individual gene, the value for undifferentiated WT ES cells was set to 1. NDST1 and -2 were not assayed for in the mutant cells. Error bars , S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Undersulfation of Heparan Sulfate Restricts Differentiation Potential of Mouse Embryonic Stem Cells *

    doi: 10.1074/jbc.M111.337030

    Figure Lengend Snippet: Gene expression analysis of WT and NDST1 −/− NDST2 −/− ES cells, before and after 6 days of neural differentiation. WT and mutant ES cells were cultured in ES cell medium ( Undiff ) or for 6 days in N2B27 medium to promote neural differentiation ( Diff ). Hypoxanthine-guanine phosphoribosyltransferase ( HPRT ) was used as an internal control ( A , top ). A–C , semiquantitative RT-PCR analysis. A , expression of the undifferentiated markers Oct3/4 and nanog, neural precursor protein nestin, mesoderm marker Brachyury, and primitive endoderm marker GATA4. B , expression of BMP4 and the BMP inhibitors noggin, chordin, and follistatin. C , expression of FGF4, FGF5, and the FGF receptors 1b, 1c, 2b, 2c, 3, and 4. D , real-time RT-PCR analysis of NDST1, -2, -3, and -4 transcripts. For each individual gene, the value for undifferentiated WT ES cells was set to 1. NDST1 and -2 were not assayed for in the mutant cells. Error bars , S.D.

    Article Snippet: Primary antibodies were Oct3/4 (1:100; Santa Cruz Biotechnology, Inc.), nestin (1:200; Chemicon), β-III-tubulin (Tuj1, 1:500; Covance), and glial fibrillary acidic protein (GFAP) (1:200; Sigma-Aldrich).

    Techniques: Expressing, Mutagenesis, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Marker, Quantitative RT-PCR

    Immunohistochemical staining for OCT3/4 and SOX2 in human prostate cancer tissues. A , representative results of at least 2 independent experiments reveal immunostaining for OCT3/4 and SOX2 using prostate tissue arrays. Neg ., negative. Int ., intermediate. Brown areas indicate positive nuclear staining. Reduced from ×20. Scale bar represents 70 µm. Insets, reduced from ×40. B and C , classification of different GS samples based on staining intensity. GS 5–6 and 7–8 tissues were significantly different than normal ( N ) and BPH tissue (2-tailed Mann-Whitney rank test p

    Journal: The Journal of urology

    Article Title: Expression of Pluripotent Stem Cell Reprogramming Factors by Prostate Tumor Initiating Cells

    doi: 10.1016/j.juro.2009.12.092

    Figure Lengend Snippet: Immunohistochemical staining for OCT3/4 and SOX2 in human prostate cancer tissues. A , representative results of at least 2 independent experiments reveal immunostaining for OCT3/4 and SOX2 using prostate tissue arrays. Neg ., negative. Int ., intermediate. Brown areas indicate positive nuclear staining. Reduced from ×20. Scale bar represents 70 µm. Insets, reduced from ×40. B and C , classification of different GS samples based on staining intensity. GS 5–6 and 7–8 tissues were significantly different than normal ( N ) and BPH tissue (2-tailed Mann-Whitney rank test p

    Article Snippet: Cells were blocked with 10% goat serum/0.05% Triton X-100/phosphate buffered saline before incubation with primary antibodies, including anti-E-cadherin, anti-OCT3/4 (Santa Cruz Biotechnology), anti-SOX2 (Abcam®) and anti-β-catenin (BD Bio-sciences), overnight at 4C.

    Techniques: Immunohistochemistry, Staining, Immunostaining, MANN-WHITNEY

    Representative results show identification of stem cell-like tumor cells with pluripotent stem cell reprogramming factors in 3 independent experiments in prostate cancer cell lines. A and B , RT-PCR or Western blot detected OCT3/4, SOX2, Nanog, c-Myc and Klf4 expression in DU145 and PC3 cell lines with human embryonic stem cells ( hESC ) as control. Data were normalized to β-actin. C , DU145 and PC3 cells were immunostained for OCT3/4 (red areas), SOX2 (green areas) and DAPI (blue areas). Arrow indicates merged OCT3/4 and SOX2 staining ( Merge ). Scale bar represents 20 µm. Reduced from ×40.

    Journal: The Journal of urology

    Article Title: Expression of Pluripotent Stem Cell Reprogramming Factors by Prostate Tumor Initiating Cells

    doi: 10.1016/j.juro.2009.12.092

    Figure Lengend Snippet: Representative results show identification of stem cell-like tumor cells with pluripotent stem cell reprogramming factors in 3 independent experiments in prostate cancer cell lines. A and B , RT-PCR or Western blot detected OCT3/4, SOX2, Nanog, c-Myc and Klf4 expression in DU145 and PC3 cell lines with human embryonic stem cells ( hESC ) as control. Data were normalized to β-actin. C , DU145 and PC3 cells were immunostained for OCT3/4 (red areas), SOX2 (green areas) and DAPI (blue areas). Arrow indicates merged OCT3/4 and SOX2 staining ( Merge ). Scale bar represents 20 µm. Reduced from ×40.

    Article Snippet: Cells were blocked with 10% goat serum/0.05% Triton X-100/phosphate buffered saline before incubation with primary antibodies, including anti-E-cadherin, anti-OCT3/4 (Santa Cruz Biotechnology), anti-SOX2 (Abcam®) and anti-β-catenin (BD Bio-sciences), overnight at 4C.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Staining

    Isolation of stem cell-like prostate tumor cells. A , to identify surface markers for isolating stem cell-like cells from prostate cell lines DU145 and PC3 cells were immunostained for OCT3/4 (red areas), E-cadherin ( Ecad ) (green areas) and DAPI (blue areas). OCT3/4 and E-cadherin staining was also merged (arrow). Reduced from ×40. Scale bar represents 20 µm. B and C , for phenotypic analysis of DU145 and PC3 cells using double staining with E-cadherin and CD44 or integrin-α2β1 cells were gated on E-cadherin + (green curves) or E-cadherin − (blue curves) population. FITC , fluorescein isothiocyanate. D and E , flow cytometry ( FCS ) analysis of DU145 and PC3 cells reveals E-cadherin ( E-cad ) expression. Isotype matched controls were used to set analysis gates for E-cadherin cell sorting. F , representative results of 3 independent experiments show that RT-PCR identified OCT3/4, SOX2, Nanog, c-Myc and Klf4 expression in E-cadherin + and E-cadherin − cells isolated from DU145 and PC3 cells. Data were normalized to β-actin.

    Journal: The Journal of urology

    Article Title: Expression of Pluripotent Stem Cell Reprogramming Factors by Prostate Tumor Initiating Cells

    doi: 10.1016/j.juro.2009.12.092

    Figure Lengend Snippet: Isolation of stem cell-like prostate tumor cells. A , to identify surface markers for isolating stem cell-like cells from prostate cell lines DU145 and PC3 cells were immunostained for OCT3/4 (red areas), E-cadherin ( Ecad ) (green areas) and DAPI (blue areas). OCT3/4 and E-cadherin staining was also merged (arrow). Reduced from ×40. Scale bar represents 20 µm. B and C , for phenotypic analysis of DU145 and PC3 cells using double staining with E-cadherin and CD44 or integrin-α2β1 cells were gated on E-cadherin + (green curves) or E-cadherin − (blue curves) population. FITC , fluorescein isothiocyanate. D and E , flow cytometry ( FCS ) analysis of DU145 and PC3 cells reveals E-cadherin ( E-cad ) expression. Isotype matched controls were used to set analysis gates for E-cadherin cell sorting. F , representative results of 3 independent experiments show that RT-PCR identified OCT3/4, SOX2, Nanog, c-Myc and Klf4 expression in E-cadherin + and E-cadherin − cells isolated from DU145 and PC3 cells. Data were normalized to β-actin.

    Article Snippet: Cells were blocked with 10% goat serum/0.05% Triton X-100/phosphate buffered saline before incubation with primary antibodies, including anti-E-cadherin, anti-OCT3/4 (Santa Cruz Biotechnology), anti-SOX2 (Abcam®) and anti-β-catenin (BD Bio-sciences), overnight at 4C.

    Techniques: Isolation, Staining, Double Staining, Flow Cytometry, Cytometry, Expressing, FACS, Reverse Transcription Polymerase Chain Reaction

    OCT3/4 or SOX2 knockdown tumorigenicity in DU145 and PC3 cells. A and B , Western blot shows decreased OCT3/4 or SOX2 protein levels in human DU145 and PC3 prostate cancer cells transfected with shRNA. Unsorted DU145 (1 × 10 5 ) or PC3 (3 × 10 5 ) cells were subcutaneously injected in SCID mice. C and D , mean ± SD tumor volume after OCT 3/4, SOX2 or control shRNA treatment.

    Journal: The Journal of urology

    Article Title: Expression of Pluripotent Stem Cell Reprogramming Factors by Prostate Tumor Initiating Cells

    doi: 10.1016/j.juro.2009.12.092

    Figure Lengend Snippet: OCT3/4 or SOX2 knockdown tumorigenicity in DU145 and PC3 cells. A and B , Western blot shows decreased OCT3/4 or SOX2 protein levels in human DU145 and PC3 prostate cancer cells transfected with shRNA. Unsorted DU145 (1 × 10 5 ) or PC3 (3 × 10 5 ) cells were subcutaneously injected in SCID mice. C and D , mean ± SD tumor volume after OCT 3/4, SOX2 or control shRNA treatment.

    Article Snippet: Cells were blocked with 10% goat serum/0.05% Triton X-100/phosphate buffered saline before incubation with primary antibodies, including anti-E-cadherin, anti-OCT3/4 (Santa Cruz Biotechnology), anti-SOX2 (Abcam®) and anti-β-catenin (BD Bio-sciences), overnight at 4C.

    Techniques: Western Blot, Transfection, shRNA, Injection, Mouse Assay

    GridMedian extracts features from fluorescent image data while reduce the size of data. ( A,B ) The median intensities of all 144 grids from the test image set ( Fig. 1A ) are plotted. Anti-Oct4 intensity does not correlate with EGFP intensity ( A ), while well correlate with H33342 intensity ( B ). The 144 grids are classified to 4 sub-classes by Mclust according to the 3-channel intensities (H33342, anti-Oct4 and EGFP) without drawing threshold lines arbitrarily. The 4 sub-classes are shown in 4 different colours and tentatively designated. Misclassified grids are encircled ( B ). ( C ) Kernel density estimations indicate very similar EGFP expression profiles between EGFP(−) and void sub-classes. High-peak of sub-class designated “other” (blue) corresponds cytoplasmic region of EGFP(+) mESCs. ( D ) Oct4 expression profiles between EGFP(−) and EGFP(+) sub-classes suggests higher expression of Oct4 in EGFP(+) mESCs. Smoothing kernel = Gaussian, bandwidth = 0.05. ( E ) After successful application of “Median_IQR filter” (see Supplementary Fig. S2 ) on the 144 grids, 67 of class 3 grids are again classified by Mclust leading EGFP(+) and EGFP(−) sub-classes. Suspected misclassified grids are encircled. ( F ) The Oct4 expression profiles between 51 of EGFP(+) and 16 of EGFP(−) sub-classes indicate higher expression of Oct4 in mESCs. Sample script (GBIQ_ESC.R) to reproduce the result and figures is available from https://github.com/yo-ninomy/DemoScripts .

    Journal: Scientific Reports

    Article Title: GBIQ: a non-arbitrary, non-biased method for quantification of fluorescent images

    doi: 10.1038/srep26454

    Figure Lengend Snippet: GridMedian extracts features from fluorescent image data while reduce the size of data. ( A,B ) The median intensities of all 144 grids from the test image set ( Fig. 1A ) are plotted. Anti-Oct4 intensity does not correlate with EGFP intensity ( A ), while well correlate with H33342 intensity ( B ). The 144 grids are classified to 4 sub-classes by Mclust according to the 3-channel intensities (H33342, anti-Oct4 and EGFP) without drawing threshold lines arbitrarily. The 4 sub-classes are shown in 4 different colours and tentatively designated. Misclassified grids are encircled ( B ). ( C ) Kernel density estimations indicate very similar EGFP expression profiles between EGFP(−) and void sub-classes. High-peak of sub-class designated “other” (blue) corresponds cytoplasmic region of EGFP(+) mESCs. ( D ) Oct4 expression profiles between EGFP(−) and EGFP(+) sub-classes suggests higher expression of Oct4 in EGFP(+) mESCs. Smoothing kernel = Gaussian, bandwidth = 0.05. ( E ) After successful application of “Median_IQR filter” (see Supplementary Fig. S2 ) on the 144 grids, 67 of class 3 grids are again classified by Mclust leading EGFP(+) and EGFP(−) sub-classes. Suspected misclassified grids are encircled. ( F ) The Oct4 expression profiles between 51 of EGFP(+) and 16 of EGFP(−) sub-classes indicate higher expression of Oct4 in mESCs. Sample script (GBIQ_ESC.R) to reproduce the result and figures is available from https://github.com/yo-ninomy/DemoScripts .

    Article Snippet: Anti-Oct4 (Pou5f1) (Santa Cruz, sc-5279, 1:100) with indicated dilution ratio in the blocking solution was applied for overnight at 4 °C.

    Techniques: Expressing

    GBIQ reproduces a comparative result to a standard cell-segmentation method. ( A,B ) The 903 observations after applying the “Median_IQR filter” and Mclust on the filtered dataset of 3 full resolution image sets are plotted. ( C,D ) Kernel density estimation of EGFP and Oct4 expression profiles show distinctive properties among the 3 sub-classes (red, green and blue). ( E,F ) Expression level of Oct4 is measured by TQ on the same image sets that consist 3 sets of Dox-treated fluorescent micrographs. From the 3 sets of images, TQ identifies 530 cells that are classified into 3 sub-classes (red, green and blue) by Mclust ( E ). Kernel density estimation shows TQ also reports higher expression of Oct4 in EGFP(+) mESCs ( F ). Smoothing kernel = Gaussian. ( C,D ) GBIQ, bandwidth = 0.05. ( F ) TQ, bandwidth = 10.

    Journal: Scientific Reports

    Article Title: GBIQ: a non-arbitrary, non-biased method for quantification of fluorescent images

    doi: 10.1038/srep26454

    Figure Lengend Snippet: GBIQ reproduces a comparative result to a standard cell-segmentation method. ( A,B ) The 903 observations after applying the “Median_IQR filter” and Mclust on the filtered dataset of 3 full resolution image sets are plotted. ( C,D ) Kernel density estimation of EGFP and Oct4 expression profiles show distinctive properties among the 3 sub-classes (red, green and blue). ( E,F ) Expression level of Oct4 is measured by TQ on the same image sets that consist 3 sets of Dox-treated fluorescent micrographs. From the 3 sets of images, TQ identifies 530 cells that are classified into 3 sub-classes (red, green and blue) by Mclust ( E ). Kernel density estimation shows TQ also reports higher expression of Oct4 in EGFP(+) mESCs ( F ). Smoothing kernel = Gaussian. ( C,D ) GBIQ, bandwidth = 0.05. ( F ) TQ, bandwidth = 10.

    Article Snippet: Anti-Oct4 (Pou5f1) (Santa Cruz, sc-5279, 1:100) with indicated dilution ratio in the blocking solution was applied for overnight at 4 °C.

    Techniques: Expressing

    Differentiation of ESCs. (a, b) Immunostaining images of Oct3/4 and FGF5 for ESCs grown on collagen plates (a) or E-cadherin-coated plates (b) with two types of medium: maintenance medium and differentiation medium. The nuclei were stained with DAPI ( blue ). (c) Microscopic images of bright field and Nanog-GFP fluorescence for living ESCs on E-cadherin-coated plates in maintenance medium and differentiation medium. (d) Mean GFP intensity in individual ESCs from (c) . GFP was quantified by multiplying the cell body area (pixel counts) by the average brightness (256 grayscale) (** P

    Journal: Stem Cells and Development

    Article Title: Asymmetricity Between Sister Cells of Pluripotent Stem Cells at the Onset of Differentiation

    doi: 10.1089/scd.2017.0113

    Figure Lengend Snippet: Differentiation of ESCs. (a, b) Immunostaining images of Oct3/4 and FGF5 for ESCs grown on collagen plates (a) or E-cadherin-coated plates (b) with two types of medium: maintenance medium and differentiation medium. The nuclei were stained with DAPI ( blue ). (c) Microscopic images of bright field and Nanog-GFP fluorescence for living ESCs on E-cadherin-coated plates in maintenance medium and differentiation medium. (d) Mean GFP intensity in individual ESCs from (c) . GFP was quantified by multiplying the cell body area (pixel counts) by the average brightness (256 grayscale) (** P

    Article Snippet: For immunostaining, the ESCs were fixed in 10% formaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% BSA, and then stained with an anti-Oct3/4 antibody (rabbit polyclonal IgG 1:100; Santa Cruz Biotechnology, Dallas, TX) or an anti-FGF5 antibody (rabbit polyclonal IgG 1:100; Santa Cruz Biotechnology) as primary antibodies.

    Techniques: Immunostaining, Staining, Fluorescence

    Characterization of mesenchymal stem cells. Primary human mesenchymal stem cells immunostained for Oct3/4 (A), and Nanog (B). Cells were fixed and permeabilized. Primary antibodies were detected by addition of fluorescein-labelled (FITC) (green) and Alexa Fluor 594-labelled (red) secondary antibodies. E, G-I: Double immunofluorescence staining for CD90 (E), CD105 (G, H, I) and C-X-C-chemokine receptor type 4 (CXCR4) (panels E, G, H, I) in lung tissue sections from patients with histologically confirmed IPF/UIP and chronic fibrotic hypersensitivity pneumonitis. Primary antibodies were detected by addition of secondary antibodies labelled with fluorescein FITC (green) for CD105 and CXCR4 detection or Cy3-labelled (red) for CD90 and CXCR4 detection. Magnification x40. Images were acquired using LSM 510 confocal microscope. Panel H and I is a 3D reconstruction to show more clear co stainings on the same cell. F: Co-staining with CD44 (pink) and CXCR4 (dark red) (Scale bar 2μm, Magnification x100 (oil)). (C, D) Oct3/4 (C) and Nanog (D) mRNA expression in mesenchymal stem cells (black bars) and in fibroblasts (grey bars). RNA expression was assessed by quantitative real time RT-PCR. Data are presented as mean ± SEM of independent experiments performed in three different cell lines.

    Journal: PLoS ONE

    Article Title: Multipotent mesenchymal stem cells in lung fibrosis

    doi: 10.1371/journal.pone.0181946

    Figure Lengend Snippet: Characterization of mesenchymal stem cells. Primary human mesenchymal stem cells immunostained for Oct3/4 (A), and Nanog (B). Cells were fixed and permeabilized. Primary antibodies were detected by addition of fluorescein-labelled (FITC) (green) and Alexa Fluor 594-labelled (red) secondary antibodies. E, G-I: Double immunofluorescence staining for CD90 (E), CD105 (G, H, I) and C-X-C-chemokine receptor type 4 (CXCR4) (panels E, G, H, I) in lung tissue sections from patients with histologically confirmed IPF/UIP and chronic fibrotic hypersensitivity pneumonitis. Primary antibodies were detected by addition of secondary antibodies labelled with fluorescein FITC (green) for CD105 and CXCR4 detection or Cy3-labelled (red) for CD90 and CXCR4 detection. Magnification x40. Images were acquired using LSM 510 confocal microscope. Panel H and I is a 3D reconstruction to show more clear co stainings on the same cell. F: Co-staining with CD44 (pink) and CXCR4 (dark red) (Scale bar 2μm, Magnification x100 (oil)). (C, D) Oct3/4 (C) and Nanog (D) mRNA expression in mesenchymal stem cells (black bars) and in fibroblasts (grey bars). RNA expression was assessed by quantitative real time RT-PCR. Data are presented as mean ± SEM of independent experiments performed in three different cell lines.

    Article Snippet: For Nanog (R & D systems) and Oct3/4 (Abcam, USA) stainings cells were fixed as described above, and incubated with primary antibodies over night at 4°C.

    Techniques: Double Immunofluorescence Staining, Microscopy, Staining, Expressing, RNA Expression, Quantitative RT-PCR

    Time-lapse and immunostaining images of “8”-shaped hatching. ( A ) At 119 h 30 min post-insemination. The outside blastocyst is expanded. ( B ) At 119 h 35 min post-insemination. The outside blastocyst has collapsed (arrow). ( C ) At 119 h 59 min post-insemination. The outside blastocyst has re-expanded. ( D ) Immunostaining at 120 h post-insemination, immediately after the time point shown in C . A merged image of Oct3/4 (red), Cdx2 (green), and Hoechst (blue) staining is shown. Oct3/4-positive cells (arrowhead) were attached to the TE of the outside blastocyst opposite the hatching site. Scale bar, 20 μm.

    Journal: PLoS ONE

    Article Title: The location of “8”-shaped hatching influences inner cell mass formation in mouse blastocysts

    doi: 10.1371/journal.pone.0175150

    Figure Lengend Snippet: Time-lapse and immunostaining images of “8”-shaped hatching. ( A ) At 119 h 30 min post-insemination. The outside blastocyst is expanded. ( B ) At 119 h 35 min post-insemination. The outside blastocyst has collapsed (arrow). ( C ) At 119 h 59 min post-insemination. The outside blastocyst has re-expanded. ( D ) Immunostaining at 120 h post-insemination, immediately after the time point shown in C . A merged image of Oct3/4 (red), Cdx2 (green), and Hoechst (blue) staining is shown. Oct3/4-positive cells (arrowhead) were attached to the TE of the outside blastocyst opposite the hatching site. Scale bar, 20 μm.

    Article Snippet: After blocking, embryos were incubated with anti-Oct3/4 (1:50) primary antibodies overnight at 4°C, washed thrice in wash buffer-2, and then incubated with secondary antibodies (1:200; Alexa Flour 647-conjugated donkey anti-goat, Abcam) for 30 min at room temperature.

    Techniques: Immunostaining, Staining

    Method used for determining ICM size. Oct3/4 (red) was stained with specific antibodies, and Oct3/4-positive cell masses were identified as ICM. The 3D confocal image of each blastocyst was rotated to set the ICM center on the cross-section ( A, B ), and then the ICM end-to-end distance along the curved surface ( C ) was measured by following each of the perpendicular lines (curves) on the blastocyst image. Subsequently, these measurements for each blastocyst were averaged to obtain the “width” as an indicator of the size of the ICM in the blastocyst. Scale bar, 20 μm.

    Journal: PLoS ONE

    Article Title: The location of “8”-shaped hatching influences inner cell mass formation in mouse blastocysts

    doi: 10.1371/journal.pone.0175150

    Figure Lengend Snippet: Method used for determining ICM size. Oct3/4 (red) was stained with specific antibodies, and Oct3/4-positive cell masses were identified as ICM. The 3D confocal image of each blastocyst was rotated to set the ICM center on the cross-section ( A, B ), and then the ICM end-to-end distance along the curved surface ( C ) was measured by following each of the perpendicular lines (curves) on the blastocyst image. Subsequently, these measurements for each blastocyst were averaged to obtain the “width” as an indicator of the size of the ICM in the blastocyst. Scale bar, 20 μm.

    Article Snippet: After blocking, embryos were incubated with anti-Oct3/4 (1:50) primary antibodies overnight at 4°C, washed thrice in wash buffer-2, and then incubated with secondary antibodies (1:200; Alexa Flour 647-conjugated donkey anti-goat, Abcam) for 30 min at room temperature.

    Techniques: Staining

    Time-lapse and immunostaining images of a U-shaped-hatching blastocyst that hatched completely, in which an initial “8”-shaped hatching occurred near the ICM. ( A ) At 90 h 45 min after insemination. Near the ICM (surrounded by arrowheads), an “8”-shaped hatch appears (arrow). ( B ) At 117 h 57 min after insemination. U-shaped hatching appears (arrow) in this embryo. ( C ) At 119 h 47 min after insemination. The embryo has hatched completely. ( D ) Immunostaining at 120 h post-insemination. The result confirmed that the ICM, stained by anti-Oct3/4 (red), is scattered. Scale bar, 20 μm.

    Journal: PLoS ONE

    Article Title: The location of “8”-shaped hatching influences inner cell mass formation in mouse blastocysts

    doi: 10.1371/journal.pone.0175150

    Figure Lengend Snippet: Time-lapse and immunostaining images of a U-shaped-hatching blastocyst that hatched completely, in which an initial “8”-shaped hatching occurred near the ICM. ( A ) At 90 h 45 min after insemination. Near the ICM (surrounded by arrowheads), an “8”-shaped hatch appears (arrow). ( B ) At 117 h 57 min after insemination. U-shaped hatching appears (arrow) in this embryo. ( C ) At 119 h 47 min after insemination. The embryo has hatched completely. ( D ) Immunostaining at 120 h post-insemination. The result confirmed that the ICM, stained by anti-Oct3/4 (red), is scattered. Scale bar, 20 μm.

    Article Snippet: After blocking, embryos were incubated with anti-Oct3/4 (1:50) primary antibodies overnight at 4°C, washed thrice in wash buffer-2, and then incubated with secondary antibodies (1:200; Alexa Flour 647-conjugated donkey anti-goat, Abcam) for 30 min at room temperature.

    Techniques: Immunostaining, Staining

    Immunostaining of “8”-shaped-hatching and U-shaped-hatching blastocysts. Cdx2 (green) and Oct3/4 (red) were stained with specific antibodies, and nuclei were stained with Hoechst (blue). In the case of “8”-shaped hatching, the figure shows embryos in the Near group ( A–D ) and Far group ( E–H ). Whereas the ICM tended to be morphologically compact in the Far group ( G ), it was scattered in the Near group ( C ; see Fig 7C ).

    Journal: PLoS ONE

    Article Title: The location of “8”-shaped hatching influences inner cell mass formation in mouse blastocysts

    doi: 10.1371/journal.pone.0175150

    Figure Lengend Snippet: Immunostaining of “8”-shaped-hatching and U-shaped-hatching blastocysts. Cdx2 (green) and Oct3/4 (red) were stained with specific antibodies, and nuclei were stained with Hoechst (blue). In the case of “8”-shaped hatching, the figure shows embryos in the Near group ( A–D ) and Far group ( E–H ). Whereas the ICM tended to be morphologically compact in the Far group ( G ), it was scattered in the Near group ( C ; see Fig 7C ).

    Article Snippet: After blocking, embryos were incubated with anti-Oct3/4 (1:50) primary antibodies overnight at 4°C, washed thrice in wash buffer-2, and then incubated with secondary antibodies (1:200; Alexa Flour 647-conjugated donkey anti-goat, Abcam) for 30 min at room temperature.

    Techniques: Immunostaining, Staining

    A, quantification of hOCT3 and hPMAT mRNA levels in stably transfected Flp-in HEK293 cells. Total RNA from Flp-293-pcDNA5, hOCT3, and hPMAT cells were extracted and reverse-transcribed. hOCT3 and hPMAT mRNA copy numbers were determined by Taqman real-time RT-PCR using specific primers and probes. Diluted hOCT3 and hPMAT expression vectors with known copy numbers were used as standards to determine absolute mRNA copy numbers. B, localization of hOCT3 and hPMAT in Flp-in HEK293 cells stably transfected with hPMAT or hOCT3. Cells transfected with pcDNA5 vector were used as control. Confluent cells were immunostained with anti-OCT3 or anti-PMAT primary antibodies and Alexa Fluor-conjugated secondary antibodies. Images were taken under a fluorescent microscope with corresponding filters.

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Selective Transport of Monoamine Neurotransmitters by Human Plasma Membrane Monoamine Transporter and Organic Cation Transporter 3 S⃞

    doi: 10.1124/jpet.110.170142

    Figure Lengend Snippet: A, quantification of hOCT3 and hPMAT mRNA levels in stably transfected Flp-in HEK293 cells. Total RNA from Flp-293-pcDNA5, hOCT3, and hPMAT cells were extracted and reverse-transcribed. hOCT3 and hPMAT mRNA copy numbers were determined by Taqman real-time RT-PCR using specific primers and probes. Diluted hOCT3 and hPMAT expression vectors with known copy numbers were used as standards to determine absolute mRNA copy numbers. B, localization of hOCT3 and hPMAT in Flp-in HEK293 cells stably transfected with hPMAT or hOCT3. Cells transfected with pcDNA5 vector were used as control. Confluent cells were immunostained with anti-OCT3 or anti-PMAT primary antibodies and Alexa Fluor-conjugated secondary antibodies. Images were taken under a fluorescent microscope with corresponding filters.

    Article Snippet: After incubating with primary antibodies, cells were washed three times with DPBS containing 0.05% Triton X-100 and incubated with fluorescence-conjugated secondary antibodies for 1 h (Alexa Fluor 488 goat anti-rabbit for PMAT, Alexa Fluor 555 donkey anti-goat for OCT3, both from Invitrogen, and diluted 1:1000 in blocking buffer).

    Techniques: Stable Transfection, Transfection, Quantitative RT-PCR, Expressing, Plasmid Preparation, Microscopy

    TESC regulates the CSC-like self-renewal properties in lung cancer cells. ( A ) Clonogenicities of TESC -knockdown A549 cells with siRNA and TESC -overexpressing H460 cells transfected with expression vector ( TESC -pcDNA3.1) were examined in vitro by colony formation assay. ( B ) Cellular levels of TESC and CSC markers including CD44, CD133, Oct3/4, Sox2, and β-catenin in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( C ) RT-PCR analysis of ALDH1 isozymes in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( D ) Changes in sphere-forming capacity in TESC -knockdown A549 cells or TESC -overexpressing H460 cells. ( E ) Changes in cellular ALDH1 levels were examined using ALDEFLUOR assay in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. Data represent mean ± SD of three independent experiments using two-tailed t-test. *P

    Journal: Scientific Reports

    Article Title: Tescalcin/c-Src/IGF1Rβ-mediated STAT3 activation enhances cancer stemness and radioresistant properties through ALDH1

    doi: 10.1038/s41598-018-29142-x

    Figure Lengend Snippet: TESC regulates the CSC-like self-renewal properties in lung cancer cells. ( A ) Clonogenicities of TESC -knockdown A549 cells with siRNA and TESC -overexpressing H460 cells transfected with expression vector ( TESC -pcDNA3.1) were examined in vitro by colony formation assay. ( B ) Cellular levels of TESC and CSC markers including CD44, CD133, Oct3/4, Sox2, and β-catenin in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( C ) RT-PCR analysis of ALDH1 isozymes in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( D ) Changes in sphere-forming capacity in TESC -knockdown A549 cells or TESC -overexpressing H460 cells. ( E ) Changes in cellular ALDH1 levels were examined using ALDEFLUOR assay in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. Data represent mean ± SD of three independent experiments using two-tailed t-test. *P

    Article Snippet: Western blot analyses were performed with primary antibodies against antigens as followings: TESC (109444, Santa Cruz), phospho-IGF1Rβ (101703, Santa Cruz), IGF1Rβ (713, Santa Cruz), STAT3 (8019, Santa Cruz), phospho-STAT3 (8059, Santa Cruz), c-Src (130124, Santa Cruz), phospho-c-Src (81521, Santa Cruz), Zeb1 (25388, Santa Cruz), β-catenin (7963, Santa Cruz), GAPDH (365062, Santa Cruz), FAK (3285, Cell Signaling), phospho-FAK (8556, Cell Signaling), CD44 (5640, Cell Signaling), E-cadherin (3195, Cell Signaling), Sox-2 (3579, Cell Signaling), N-cadherin (610921, BD Biosciences), Oct3/4 (4305, Millipore), Vimentin (16409, Thermo Fisher), ALDH1A1 (52492, Abcam), ALDH1A3 (80176, Abcam), and β-actin (3700, Cell Signaling).

    Techniques: Transfection, Expressing, Plasmid Preparation, In Vitro, Colony Assay, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    Effect of Cltc Knockdown in mESCs Can Be Partially Rescued by Inhibiting TGF-β Signaling (A) Bright-field micrographs showing AP staining and morphology of Cltc knockdown mESCs in the presence of TGF-β inhibitors RepSox or SB431542 (SB). Scale bar, 50 μm. (B) Immunofluorescence micrographs showing OCT3/4 and E-CAD staining upon RepSox or SB431542 (SB) treatment in mESCs with Cltc knockdown. Scale bar, 50 μm. (C) Bar graph showing the quantitation of OCT3/4 and E-CAD-positive mESC colonies upon RepSox or SB431542 (SB) treatment in Cltc knockdown mESCs. Error bars represent mean ± SD from three independent experiments (n = 3). ∗∗ p

    Journal: Stem Cell Reports

    Article Title: Clathrin-Mediated Endocytosis Regulates a Balance between Opposing Signals to Maintain the Pluripotent State of Embryonic Stem Cells

    doi: 10.1016/j.stemcr.2018.11.018

    Figure Lengend Snippet: Effect of Cltc Knockdown in mESCs Can Be Partially Rescued by Inhibiting TGF-β Signaling (A) Bright-field micrographs showing AP staining and morphology of Cltc knockdown mESCs in the presence of TGF-β inhibitors RepSox or SB431542 (SB). Scale bar, 50 μm. (B) Immunofluorescence micrographs showing OCT3/4 and E-CAD staining upon RepSox or SB431542 (SB) treatment in mESCs with Cltc knockdown. Scale bar, 50 μm. (C) Bar graph showing the quantitation of OCT3/4 and E-CAD-positive mESC colonies upon RepSox or SB431542 (SB) treatment in Cltc knockdown mESCs. Error bars represent mean ± SD from three independent experiments (n = 3). ∗∗ p

    Article Snippet: The following antibodies were used at the appropriate concentrations: CLTC (sc-6579, Santa Cruz, goat), TGF-βR1 (sc-398, Santa Cruz, rabbit), NANOG (sc-30328, Santa Cruz, goat), SMAD2/3 (sc-7960, Santa Cruz, mouse), RAB7 (sc-81922, Santa Cruz, rabbit), OCT3/4 (no. 2849, Cell Signaling Technology, rabbit), E-CAD (no. 3195, Cell Signaling Technology, rabbit), pSMAD2/3 (no. 8828, Cell Signaling Technology, rabbit), SOX2 (ab97951, Abcam, rabbit), E-CAD (no. 610182, BD Transduction Laboratories, mouse), RAB11 (no. 610657, BD Transduction Laboratories, mouse), CLTC (no. 610500, BD Transduction Laboratories, mouse), TUBULIN (T5168, Sigma, mouse), RAB5 (no. 3547, Cell Signaling Technology, rabbit), RAB7 (no. 9367, Cell Signaling Technology, rabbit), GAPDH (no. SC25778, Santa Cruz, rabbit), Na,K+ ATPASE (no. 21713, Santa Cruz, goat), LC3 1/2 (no. 12741, Cell Signaling Technology, rabbit), LAMP2 (ab13542, Abcam, rat).

    Techniques: Staining, Immunofluorescence, Quantitation Assay

    Generation of human reprogrammed cells from fibroblasts of control healthy subjects and HNF1A MODY patients. (A). Morphology of cells from healthy subject before (day 0) and 13 days after transduction (day 13) with hSTEMCCA lentiviral vectors. Colonies resembling human embryonic stem cells, visible at day 13, were further expanded (expanded colony, shown at passage 2). GFP - expression of GFP in human primary fibroblast isolated from HNF1A MODY patients, 72 after transduction with control (FUGW) lentiviral vectors. Representative pictures (scale bar – 100 μm). (B). Analysis of hSTEMCCA construct silencing in established control (effective silencing in Ctr Repro 2 cell line) and HNF1A MODY reprogrammed cells (effective silencing in HNF1A MODY Repro 1 cell line). RT-PCR. (C,D). Immunofluorescence staining of pluripotency markers: OCT3/4A, SSEA4, TRA-1-60, TRA-1-81 in control reprogrammed cells (C) and HNF1A MODY reprogrammed cells (D). Representative pictures (scale bar – 100 μm).

    Journal: Scientific Reports

    Article Title: Induced pluripotent stem cells as a model for diabetes investigation

    doi: 10.1038/srep08597

    Figure Lengend Snippet: Generation of human reprogrammed cells from fibroblasts of control healthy subjects and HNF1A MODY patients. (A). Morphology of cells from healthy subject before (day 0) and 13 days after transduction (day 13) with hSTEMCCA lentiviral vectors. Colonies resembling human embryonic stem cells, visible at day 13, were further expanded (expanded colony, shown at passage 2). GFP - expression of GFP in human primary fibroblast isolated from HNF1A MODY patients, 72 after transduction with control (FUGW) lentiviral vectors. Representative pictures (scale bar – 100 μm). (B). Analysis of hSTEMCCA construct silencing in established control (effective silencing in Ctr Repro 2 cell line) and HNF1A MODY reprogrammed cells (effective silencing in HNF1A MODY Repro 1 cell line). RT-PCR. (C,D). Immunofluorescence staining of pluripotency markers: OCT3/4A, SSEA4, TRA-1-60, TRA-1-81 in control reprogrammed cells (C) and HNF1A MODY reprogrammed cells (D). Representative pictures (scale bar – 100 μm).

    Article Snippet: The following primary antibodies were used: Oct3/4A (1:200, Santa Cruz Biotechnology), Nanog (1:200, Abcam), SSEA-1 (1:200, Millipore), SSEA-4, TRA-1-60, TRA-1-81 (1:100, Millipore), Nestin, Vimentin (1:200, Abcam), Neurofilament heavy chain (1:1000, Abcam), smooth muscle actin (1:100, Abcam), GATA-4 (1:50 for human and 1:200 for murine cells, Santa Cruz Biotechnology), Nkx2.5 (1:200, Santa Cruz Biotechnology), vWF (1:100, Abcam), Flt-1 (1:100, Abcam), phospho eNOS (1:100, Abcam).

    Techniques: Transduction, Expressing, Isolation, Construct, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining

    Generation of wild type (WT) and Lep db/db (db/db) iPS cells. (A). Morphology of WT and db/db iPS cells. Representative pictures (scale bar – 100 μm). (B). Expression of pluripotency markers: Oct3/4A, Nanog and Sox2 in control and db/db iPSCs. Quantitative RT-PCR analysis. N = 3. (C). Immunofluorescence staining of pluripotency markers: Oct3/4A, Nanog, SSEA1 and CDy1 in WT and db/db iPS cells (scale bar – 100 μm). (D). Expression of alkaline phosphatase in WT and db/db iPS cells. Representative pictures (scale bar – 100 μm). (E). Expression of genes associated with endothelial lineage development: HO-1, proteoglycan-4 and angiotensinogen. Quantitative RT-PCR analysis. N = 2. EF2 gene was used as housekeeping gene. Each bar represents mean + SEM. The numerical data presented in Fig. 1B and 1E were not statistically significant.

    Journal: Scientific Reports

    Article Title: Induced pluripotent stem cells as a model for diabetes investigation

    doi: 10.1038/srep08597

    Figure Lengend Snippet: Generation of wild type (WT) and Lep db/db (db/db) iPS cells. (A). Morphology of WT and db/db iPS cells. Representative pictures (scale bar – 100 μm). (B). Expression of pluripotency markers: Oct3/4A, Nanog and Sox2 in control and db/db iPSCs. Quantitative RT-PCR analysis. N = 3. (C). Immunofluorescence staining of pluripotency markers: Oct3/4A, Nanog, SSEA1 and CDy1 in WT and db/db iPS cells (scale bar – 100 μm). (D). Expression of alkaline phosphatase in WT and db/db iPS cells. Representative pictures (scale bar – 100 μm). (E). Expression of genes associated with endothelial lineage development: HO-1, proteoglycan-4 and angiotensinogen. Quantitative RT-PCR analysis. N = 2. EF2 gene was used as housekeeping gene. Each bar represents mean + SEM. The numerical data presented in Fig. 1B and 1E were not statistically significant.

    Article Snippet: The following primary antibodies were used: Oct3/4A (1:200, Santa Cruz Biotechnology), Nanog (1:200, Abcam), SSEA-1 (1:200, Millipore), SSEA-4, TRA-1-60, TRA-1-81 (1:100, Millipore), Nestin, Vimentin (1:200, Abcam), Neurofilament heavy chain (1:1000, Abcam), smooth muscle actin (1:100, Abcam), GATA-4 (1:50 for human and 1:200 for murine cells, Santa Cruz Biotechnology), Nkx2.5 (1:200, Santa Cruz Biotechnology), vWF (1:100, Abcam), Flt-1 (1:100, Abcam), phospho eNOS (1:100, Abcam).

    Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

    LSD1 is minimally expressed in hiPSCs but strongly expressed in hiPSC-derived teratoma (A) We isolated whole cell lysates from normal human fibroblasts, hiPSC line 201B, 201B-derived teratoma, normal human T-lymphocytes, and cancer cell lines (K562, HeLa, and HEK293) for immunoblot analyses to determine the expression of HDAC1, HDAC3, HDAC6, LSD1, Oct3/4, and GAPDH (internal control) (upper panel). The signal intensities of each band were quantified, normalized to those of the corresponding GAPDH, and shown as relative values setting fibroblast at 1.0 (lower panel). Immunoblotting was carried out using the Simple Western System Wes. (B) We isolated total cellular RNAs and subjected them to qPCR to evaluate the expression of HDAC1, HDAC3, HDAC6 , and LSD1 mRNA. Data were quantified by the 2 –ΔΔCt method using simultaneously amplified GAPDH as a reference and are shown as relative values setting fibroblast at 1.0. (C) Frozen continuous sections were prepared from the developed teratomas and subjected to hematoxylin-eosin (HE) and immunofluorescent chemical (IFC) staining. IFC specimens were stained with anti-HDAC1, anti-HDAC6, or anti-LSD1 antibodies, followed by staining with Alexa Fluor 488-conjugated anti-rabbit IgG (green). Nuclei were counterstained with DAPI (blue). Only merged images are shown. Scale bars indicate 200 μm (upper panels) and 50 μm (lower panels), respectively. Data shown are representative of multiple independent experiments.

    Journal: Oncotarget

    Article Title: Lysine-specific demethylase 1 inhibitors prevent teratoma development from human induced pluripotent stem cells

    doi: 10.18632/oncotarget.24030

    Figure Lengend Snippet: LSD1 is minimally expressed in hiPSCs but strongly expressed in hiPSC-derived teratoma (A) We isolated whole cell lysates from normal human fibroblasts, hiPSC line 201B, 201B-derived teratoma, normal human T-lymphocytes, and cancer cell lines (K562, HeLa, and HEK293) for immunoblot analyses to determine the expression of HDAC1, HDAC3, HDAC6, LSD1, Oct3/4, and GAPDH (internal control) (upper panel). The signal intensities of each band were quantified, normalized to those of the corresponding GAPDH, and shown as relative values setting fibroblast at 1.0 (lower panel). Immunoblotting was carried out using the Simple Western System Wes. (B) We isolated total cellular RNAs and subjected them to qPCR to evaluate the expression of HDAC1, HDAC3, HDAC6 , and LSD1 mRNA. Data were quantified by the 2 –ΔΔCt method using simultaneously amplified GAPDH as a reference and are shown as relative values setting fibroblast at 1.0. (C) Frozen continuous sections were prepared from the developed teratomas and subjected to hematoxylin-eosin (HE) and immunofluorescent chemical (IFC) staining. IFC specimens were stained with anti-HDAC1, anti-HDAC6, or anti-LSD1 antibodies, followed by staining with Alexa Fluor 488-conjugated anti-rabbit IgG (green). Nuclei were counterstained with DAPI (blue). Only merged images are shown. Scale bars indicate 200 μm (upper panels) and 50 μm (lower panels), respectively. Data shown are representative of multiple independent experiments.

    Article Snippet: We carried out immunoblotting using the Wes™ Simple Western System (Protein Simple, San Jose, CA) and specific antibodies against HDAC1-8 (Bethyl Laboratories Inc, Montgomery, TX), LSD1 (Cell Signaling Technology, Beverly, MA), Oct3/4 (Invitrogen), ßIII-tubulin, SOX17, ɑ-smooth muscle actin (Gene Tex Inc, Irvine, CA), di-methylated H3K4 (Active Motif, Carlsbad, CA), di-methylated H3K9 (Millipore), and GAPDH (Cell Signaling Technology).

    Techniques: Derivative Assay, Isolation, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Amplification, Staining

    Metformin decreases K7M2 xenograft tumor growth and inhibits lung metastasis in vivo . (a) Metformin suppressed tumorigenicity of K7M2 OSCs in vivo by limited dilution assay. n = 7. (b) The tumor formation rate of K7M2 OSCs treated with metformin. (c) The tumor volumes and (d) tumor weights at the end of the experiments. (e) Images of resected tumor xenografts on day 21. n = 6. (f) The tumor volumes and (g) the tumor weights at the end of the experiments. (h) Numbers of mice with lung metastasis. (i) H E staining of lung tissues for metastatic nodules. The arrows represent smaller tumor nodules in the lung. Scale bars = 100 μ m. (j) Immunohistochemical staining of LC3, ATG5, Ki67, Sox2, and Oct4 of three different tumors from each group. Scale bars = 100 μ m. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Metformin Suppresses Self-Renewal Ability and Tumorigenicity of Osteosarcoma Stem Cells via Reactive Oxygen Species-Mediated Apoptosis and Autophagy

    doi: 10.1155/2019/9290728

    Figure Lengend Snippet: Metformin decreases K7M2 xenograft tumor growth and inhibits lung metastasis in vivo . (a) Metformin suppressed tumorigenicity of K7M2 OSCs in vivo by limited dilution assay. n = 7. (b) The tumor formation rate of K7M2 OSCs treated with metformin. (c) The tumor volumes and (d) tumor weights at the end of the experiments. (e) Images of resected tumor xenografts on day 21. n = 6. (f) The tumor volumes and (g) the tumor weights at the end of the experiments. (h) Numbers of mice with lung metastasis. (i) H E staining of lung tissues for metastatic nodules. The arrows represent smaller tumor nodules in the lung. Scale bars = 100 μ m. (j) Immunohistochemical staining of LC3, ATG5, Ki67, Sox2, and Oct4 of three different tumors from each group. Scale bars = 100 μ m. ∗ P

    Article Snippet: Alexa Fluor® 488 mouse/human anti-Sox2 antibody (656109), PE anti-mouse/human Oct4 antibody (653703), APC anti-mouse/human CD44 antibody (103011), APC anti-mouse CD133 antibody (141207), and PE/Cy7 anti-human CD133 antibody (372809) were obtained from Biolegend (San Diego, USA).

    Techniques: In Vivo, Dilution Assay, Mouse Assay, Staining, Immunohistochemistry

    Characteristics of OSCs. (a) The representative images of SP cells from K7M2 and MG63 osteosarcoma cell lines. The median value of K7M2 and MG63 SP cells was 1.25% and 1.07%, respectively. n = 5. (b) The representative TEM images of autophagosomes in K7M2 and MG63 SP cells. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (c) Tumor spheres of K7M2 and MG63 osteosarcoma cells after culturing in the serum-free medium DMEM/F12-bFGF-EGF-B27 for 7 days. The parental K7M2 and MG63 cells cultured in DMEM/F12 supplemented with 1% FBS served as a control. Scale bars = 50 μ m. n = 5. (d) Western blot analysis of the pluripotent transcription factors Sox2, Oct4, and Nanog and the autophagy markers LC3, ATG5, and ATG7 in K7M2 and MG63 OSCs. Data are shown as mean ± SD, n = 3. (e) The mRNA expression levels of the pluripotency-associated genes SOX2 , OCT4 , and NANOG and the autophagy-related genes ATG5 and ATG7 . n = 3. (f) Immunofluorescence analysis of autophagy in K7M2 and MG63 SP cells. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 200 μ m. n = 3. (g) Osteogenic and chondrogenic differentiation of K7M2 and MG63 SP cells. Cells differentiated into osteoblasts and chondroblasts were detected by staining with Alizarin Red and Alcian Blue. Scale bars = 100 μ m. n = 3. (h) Flow cytometry-based assay for the pluripotent transcription factors Sox2 and Oct4 and the CSC surface markers CD44, CD105, CD133, and Stro-1 in K7M2 and MG63 SP cells. n = 3. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Metformin Suppresses Self-Renewal Ability and Tumorigenicity of Osteosarcoma Stem Cells via Reactive Oxygen Species-Mediated Apoptosis and Autophagy

    doi: 10.1155/2019/9290728

    Figure Lengend Snippet: Characteristics of OSCs. (a) The representative images of SP cells from K7M2 and MG63 osteosarcoma cell lines. The median value of K7M2 and MG63 SP cells was 1.25% and 1.07%, respectively. n = 5. (b) The representative TEM images of autophagosomes in K7M2 and MG63 SP cells. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (c) Tumor spheres of K7M2 and MG63 osteosarcoma cells after culturing in the serum-free medium DMEM/F12-bFGF-EGF-B27 for 7 days. The parental K7M2 and MG63 cells cultured in DMEM/F12 supplemented with 1% FBS served as a control. Scale bars = 50 μ m. n = 5. (d) Western blot analysis of the pluripotent transcription factors Sox2, Oct4, and Nanog and the autophagy markers LC3, ATG5, and ATG7 in K7M2 and MG63 OSCs. Data are shown as mean ± SD, n = 3. (e) The mRNA expression levels of the pluripotency-associated genes SOX2 , OCT4 , and NANOG and the autophagy-related genes ATG5 and ATG7 . n = 3. (f) Immunofluorescence analysis of autophagy in K7M2 and MG63 SP cells. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 200 μ m. n = 3. (g) Osteogenic and chondrogenic differentiation of K7M2 and MG63 SP cells. Cells differentiated into osteoblasts and chondroblasts were detected by staining with Alizarin Red and Alcian Blue. Scale bars = 100 μ m. n = 3. (h) Flow cytometry-based assay for the pluripotent transcription factors Sox2 and Oct4 and the CSC surface markers CD44, CD105, CD133, and Stro-1 in K7M2 and MG63 SP cells. n = 3. ∗ P

    Article Snippet: Alexa Fluor® 488 mouse/human anti-Sox2 antibody (656109), PE anti-mouse/human Oct4 antibody (653703), APC anti-mouse/human CD44 antibody (103011), APC anti-mouse CD133 antibody (141207), and PE/Cy7 anti-human CD133 antibody (372809) were obtained from Biolegend (San Diego, USA).

    Techniques: Transmission Electron Microscopy, Cell Culture, Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry, Cytometry

    Autophagy regulates the homeostasis of pluripotency in OSCs. (a) TEM images of autophagosomes in metformin-treated K7M2 and MG63 OSCs. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (b) Immunofluorescence analysis of autophagy in K7M2 and MG63 OSCs. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 50 μ m. n = 3. (c) Flow cytometry analysis of the effect of autophagy on ALDH1 activity. The upper panel shows that OSCs were incubated with DEAB, a specific ALDH inhibitor. The lower panel shows ALDH1-positive OSCs. n = 3. (d) The percentage of ALDH1-positive OSCs. (e) Clone formation of K7M2 and MG63 OSCs. Scale bars = 50 μ m. n = 5. (f) SEM images of sphere forming of K7M2 and MG63 OSCs. Scale bars = 20 μ m. n = 3. (g) Western blot analysis of the autophagy markers ATG5 and ATG7 and the pluripotency-associated proteins Sox2 and Oct4 in K7M2 and MG63 OSCs. n = 3. (h) Densitometric analyses of autophagy markers and pluripotency-associated proteins. Each experiment was conducted at least three occasions. ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Metformin Suppresses Self-Renewal Ability and Tumorigenicity of Osteosarcoma Stem Cells via Reactive Oxygen Species-Mediated Apoptosis and Autophagy

    doi: 10.1155/2019/9290728

    Figure Lengend Snippet: Autophagy regulates the homeostasis of pluripotency in OSCs. (a) TEM images of autophagosomes in metformin-treated K7M2 and MG63 OSCs. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (b) Immunofluorescence analysis of autophagy in K7M2 and MG63 OSCs. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 50 μ m. n = 3. (c) Flow cytometry analysis of the effect of autophagy on ALDH1 activity. The upper panel shows that OSCs were incubated with DEAB, a specific ALDH inhibitor. The lower panel shows ALDH1-positive OSCs. n = 3. (d) The percentage of ALDH1-positive OSCs. (e) Clone formation of K7M2 and MG63 OSCs. Scale bars = 50 μ m. n = 5. (f) SEM images of sphere forming of K7M2 and MG63 OSCs. Scale bars = 20 μ m. n = 3. (g) Western blot analysis of the autophagy markers ATG5 and ATG7 and the pluripotency-associated proteins Sox2 and Oct4 in K7M2 and MG63 OSCs. n = 3. (h) Densitometric analyses of autophagy markers and pluripotency-associated proteins. Each experiment was conducted at least three occasions. ∗ P

    Article Snippet: Alexa Fluor® 488 mouse/human anti-Sox2 antibody (656109), PE anti-mouse/human Oct4 antibody (653703), APC anti-mouse/human CD44 antibody (103011), APC anti-mouse CD133 antibody (141207), and PE/Cy7 anti-human CD133 antibody (372809) were obtained from Biolegend (San Diego, USA).

    Techniques: Transmission Electron Microscopy, Immunofluorescence, Staining, Flow Cytometry, Cytometry, Activity Assay, Incubation, Western Blot

    ECFC-derived iPSCs express typical hESC markers. (A) Immunofluorescence analysis of ECFC-derived iPSC1 line. Expression of stem cell markers: OCT3/4, SOX2, NANOG, PA, SSEA4, TRA-1-81. The cells were stained using the 488 and 546-conjugated Donkey Anti-Goat IgG or Goat Anti-Mouse IgG secondary antibody and the nuclei were counterstained with DAPI (blue). Scale bars represent 100 μm. (B) Quantitative RT-PCR analysis of the expression of endogenous (endo) and exogenous (exo) markers: OCT3/4 , SOX2 , KLF4 and C-MYC in Ctrl ECFCs, transduced ECFCs (ECFC OKSM) and ECFC-derived iPSC1 at passage 4, 7 and 10. Transcript levels were normalized to GAPDH transcript levels and relative to mean hESCs (H9 samples at P45) as a calibrator.

    Journal: PLoS ONE

    Article Title: A Novel Molecular and Functional Stemness Signature Assessing Human Cord Blood-Derived Endothelial Progenitor Cell Immaturity

    doi: 10.1371/journal.pone.0152993

    Figure Lengend Snippet: ECFC-derived iPSCs express typical hESC markers. (A) Immunofluorescence analysis of ECFC-derived iPSC1 line. Expression of stem cell markers: OCT3/4, SOX2, NANOG, PA, SSEA4, TRA-1-81. The cells were stained using the 488 and 546-conjugated Donkey Anti-Goat IgG or Goat Anti-Mouse IgG secondary antibody and the nuclei were counterstained with DAPI (blue). Scale bars represent 100 μm. (B) Quantitative RT-PCR analysis of the expression of endogenous (endo) and exogenous (exo) markers: OCT3/4 , SOX2 , KLF4 and C-MYC in Ctrl ECFCs, transduced ECFCs (ECFC OKSM) and ECFC-derived iPSC1 at passage 4, 7 and 10. Transcript levels were normalized to GAPDH transcript levels and relative to mean hESCs (H9 samples at P45) as a calibrator.

    Article Snippet: Cells were incubated overnight at +4°C with the following antibodies: anti-Phosphatase Alkaline (PA) (1:40, R & D Systems), anti-OCT3/4 (1:40, R & D Systems), anti-NANOG (1:40, R & D Systems), anti-SSEA4 (1:40, R & D Systems), anti-Tra-160/Tra1-81 (1:200, Abcam), anti-SOX2 (1:40, R & D Systems,), anti-CD31 (1:20, BD Pharmingen, clone WM59) or anti-von Willebrand factor (vWF) (1:300, Dako Cytomation A0082), diluted in 3% BSA/PBS and labeled with Alexa Fluor 488 and 546 Donkey Anti-Goat IgG or Alexa Fluor 488 and 546 Goat anti-Mouse IgG secondary antibodies (Invitrogen, Cergy-Pontoise, France).

    Techniques: Derivative Assay, Immunofluorescence, Expressing, Staining, Quantitative RT-PCR

    1α,25(OH)2D3 inhibited the stemness and impaired the mitochondrial respiration and ATP production in acidic condition. a Immunoblotting of the expression of stemness markers NESTIN, CD133, OCT4, and SOX2 in GSC2 and GSC5 cells that were treated with 1α,25(OH) 2 D 3 10 or 100 nM for 4, 8, 12, 24, and 48 h. b and c Self-renewal ability of SLCs with 1α,25(OH) 2 D 3 treatment under pH 7.4 or pH 6.8 culture conditions. Neurosphere formation assay showed the number of neurospheres (diameters larger than 50 µm) formed from GSC2 and GSC5 cells that were treated with 1α,25(OH) 2 D 3 10 or 100 nM ( b ), * P

    Journal: Cell Death & Disease

    Article Title: Acidosis enhances the self-renewal and mitochondrial respiration of stem cell-like glioma cells through CYP24A1-mediated reduction of vitamin D

    doi: 10.1038/s41419-018-1242-1

    Figure Lengend Snippet: 1α,25(OH)2D3 inhibited the stemness and impaired the mitochondrial respiration and ATP production in acidic condition. a Immunoblotting of the expression of stemness markers NESTIN, CD133, OCT4, and SOX2 in GSC2 and GSC5 cells that were treated with 1α,25(OH) 2 D 3 10 or 100 nM for 4, 8, 12, 24, and 48 h. b and c Self-renewal ability of SLCs with 1α,25(OH) 2 D 3 treatment under pH 7.4 or pH 6.8 culture conditions. Neurosphere formation assay showed the number of neurospheres (diameters larger than 50 µm) formed from GSC2 and GSC5 cells that were treated with 1α,25(OH) 2 D 3 10 or 100 nM ( b ), * P

    Article Snippet: The antibodies used included anti-CYP24A1 (Abcam, UK), anti-GFAP (Abcam, UK), anti-Tuj1 (Sigma, USA), anti-Sox2 (Abcam, UK; Abclonal, Beijing, China), anti-CD133 (Miltenyi Biotec, Germany; Abclonal, Beijing, China), anti-Nestin (Abclonal, Beijing, China), anti-Oct4 (Abclonal, Beijing, China), anti-CNPase (Abclonal, Beijing, China), and anti-β-actin (Sigma, USA).

    Techniques: Expressing, Tube Formation Assay